O gênero Nocardia é composto por bactérias gram-positivas e filamentosas, que normalmente só causam doença em indivíduos imunocomprometidos. No entanto, certo número de infecções por nocardia têm sido relatados em pacientes imunocompetentes. Nos últimos anos, observou-se um aumento da frequência de infecções por Nocardia spp. e, com o aumento do número de espécies descritas, a identificação correta tem sido de difícil obtenção mas de grande importância para a aplicação do tratamento correto e elucidação epidemiológica. O objetivo deste estudo foi caracterizar, por métodos fenotípicos e moleculares, 72 isolados de Nocardia spp. de interesse médico, avaliando as metodologias para elaborar um algoritmo de identificação. Os isolados foram provenientes da Micoteca do Instituto de Medicina Tropical de São Paulo e da rotina do Núcleo de Tuberculose e Micobacterioses do Instituto Adolfo Lutz. Os isolados foram identificados por testes fenotípicos, identificação molecular por análise de restrição (PRA-hsp65), sequenciamento dos genes hsp65 e 16S rRNA e MALDI-TOF MS (Matrix-associated laser desorption time of flight mass spectrometry). O perfil de suscetibilidade foi analisado pelo método de Concentração Inibitória Mínima (MIC) e disco difusão (DD), com os fármacos: amicacina, ciprofloxacina, minociclina, tobramicina, amoxacilina+ácido clavulânico, imipenem e sulfametoxazol+trimetoprim. Os resultados revelaram que a identificação fenotípica foi insuficiente para definir as espécies. Apenas 24 (33,4%) isolados tiveram identificação fenotípica concordante com o sequenciamento do gene 16S rRNA. Na analise feita pela técnica de PRA-hsp65 foram observados 20 (27,8%) N. brasiliensis, seis (8,3%) isolados de outras espécies de nocardias e 38 (52,8%) foram considerados novos padrões (NP). Foi detectado um isolado misto e em cinco isolados não foi obtido produto de amplificação. O sequenciamento do gene hsp65 proporcionou a identificação de 51 isolados como Nocardia, 14 foram identificados como pertencentes a outros gêneros, dos quais, um apresentou mistura de nocardia e micobactéria, sendo identificado como Mycobacterium abscessus no gene hsp65 (análise in silico) e N. otitidiscaviarum no gene 16S rRNA. Sete isolados não foram sequenciados devido à ausência de amplificação do fragmento. Os isolados analisados através do sequenciamento do gene 16S rRNA, foram identificados como Nocardia (57-79,2%), Gordonia (7-9,7%), Rhodoccocus (3-4,2%), Tsukamurella (2-2,8%), Mycobacterium (2-2,8%) e Streptomyces (1-1,3%). Para a análise de MALDI-TOF MS foi observado que, dos 72 isolados estudados, 49 foram identificados como Nocardia, 11 como pertencentes a outros gêneros e 12 amostras não puderam ser identificadas devido aos valores de leitura não serem adequados para análise. Pela primeira vez o corante resazurina foi utilizado para leitura de MIC de Nocardia sp. Entre os fármacos testados através do MIC, os que apresentaram maior sensibilidade foram amicacina (100%) e tobramicina (84%). As maiores resistências foram encontradas com os fármacos sulfametoxazol+trimetoprim (76%) e imipenem (54%). Devido à ausência de critérios interpretativos de disco difusão para o gênero Nocardia, foi elaborado um critério para o presente estudo. Os resultados obtidos no teste de DD mostraram 100% de sensibilidade para os fármacos amicacina, minociclina e sulfametoxazol+trimetroprim. Os isolados apresentaram a maior porcentagem de resistência ao fármaco ciprofloxacina (64%). Comparando os resultados de DD com os obtidos no MIC, observamos que os fármacos ciprofloxacina, imipenem e sulfametoxazol+trimetoprim apresentaram porcentagem de discordância acima de 20%. O fármaco sulfametoxazol+trimetoprim teve a maior discordância (>75%), com elevada porcentagem de isolados resistentes na MIC, mas baixa porcentagem de resistência em DD. O único fármaco com 100% de concordância entre os resultados foi amicacina. Foi elaborado um algoritmo de identificação que utiliza técnicas fenotípicas para triagem para diferenciar nocardias de outros gêneros. A identificação por PRA-hsp65 será útil na rotina de laboratório de micobactérias como identificação presuntiva. A identificação definitiva das espécies deve ser obtida pelo sequenciamento do gene 16S rRNA. / The genus Nocardia comprises filamentous gram-positive bacteria that usually cause disease only in immunocompromised individuals. However, a number of Nocardia infections have been reported in immunocompetent patients. Overall, it has been reported in the recent years an increased frequency of infections caused by Nocardia spp. due to an also increasing number of species, making the correct identification of the species more difficult. Correct identification assumes a major importance with respect to antimicrobial treatment\'s choice as well a for epidemiological investigation purposes. The objective of this study was to characterize by phenotypic and molecular methods 72 isolates of Nocardia spp. of medical interest, designing the methodologies for an identification algorithm. The isolates were obtained from the fungal collection of the Tropical Medicine Institute of São Paulo and the routine service of the Tuberculosis and Mycobacteriosis Center of the Adolfo Lutz Institute. The isolates were identified by phenotypic testing, molecular identification by restriction analysis (PRA-hsp65), hsp65 and 16S rRNA genes sequencing, and MALDI-TOF MS (matrix-associated laser desorption time-of-flight mass spectrometry). The susceptibility profile was analyzed by the Minimum Inhibitory Concentration method (MIC) and disk diffusion (DD), with the following drugs: amikacin, ciprofloxacin, minocycline, tobramycin, amoxicillin+ clavulanic acid, imipenem and sulfamethoxazole trimethoprim. The results showed that the phenotypic identification was insufficient to define the species. Only 24 (33.4%) of the isolates had phenotype identification that was concordant with the 16S rRNA gene sequencing. In the analysis made with the PRA-hsp65 technique were observed 20 (27.8%) N. brasiliensis and six (8.3%) isolates from other species of Nocardia spp., while 38 isolates (52.8%) were considered as new standards (NP). Also by this technique a mixed isolate was detected and an amplification product was not obtained in five isolates. The hsp65 gene sequencing provided identification of 51 isolates as Nocardia, 14 were identified as belonging to other genera, one of them being identified either as Mycobacterium abscessus by in silico analysis of the hsp65 gene and as N. otitidiscaviarum by the 16S rRNA gene sequencing. Seven isolates were not sequenced due to the absence of the amplified fragment. The isolates analyzed by 16S rRNA gene sequencing were identified as Nocardia (57 to 79.2%), Gordonia (7 to 9.7%), Rhodoccocus (3 to 4.2%), Tsukamurella (2-2, 8%), Mycobacterium (2 to 2.8%) and Streptomyces (1-1.3%). By MALDI-TOF MS analysis of the 72 isolates, 49 were identified as Nocardia, 11 were identified as belonging to other genera and 12 isolates could not be identified because the samples provided reading values that were inadequate for analysis. For the first time, to our knowledge, the resazurin dye was used to determine the MICs of Nocardia sp. Among the drugs tested, the most sensitives were amikacin (100%) and tobramycin (84%). The higher resistances were found with trimethoprim-sulfamethoxazole (76%) and imipenem (54%). Due to the absence of establishe criteria for the interpretation of the disk diffusion assay with Nocardia, we designed a specific criterion for this study. The results obtained in the DD test showed 100% sensitivity for the drugs amikacin, minocycline and trimethoprim-sulfamethoxazole. The isolates showed the highest percentage of resistance to ciprofloxacin drug (64%). Comparing the results with those obtained with DD and MIC, we observed that ciprofloxacin, imipenem and sulfamethoxazole-trimethoprim showed a percentage of disagreement above 20%. The sulfamethoxazole-trimethoprim drug had the highest discrepancy (> 75%), with high percentage of resistant isolates with MIC but low percentage of resistance in DD. The only drug with 100% agreement between the both results was amikacin. We designed a recognition algorithm using phenotyping techniques to screen and differentiate nocardias from other genera. The identification by PRA-hsp65 will be useful in routine mycobacteria laboratory as a presumptive identification tool. The final identification of the species should be obtained by sequencing the 16S rRNA gene.

The larger grain borer (LGB) Prostephanus truncatus (Horn) is the most important pest of farm stored maize and cassava in Africa. This alien invasive species was introduced into the continent from Mesoamerica in the late 1970s and by 2008 had spread to at least 18 countries. In contrast to indigenous primary storage pests, LGB exists as on-farm and as wild populations, hence, sustainable control must target both environments. Biological control is especially attractive for wild populations to reduce early season grain store infestation, while cultural and chemical methods are useful to protect stored produce directly. Two populations of the predator Teretrius nigrescens Lewis were introduced into several African countries' as a biocontrol agent. It has shown long-term success and cost effective control in warm-humid areas. Control has however not been successful in cool and hot-dry zones. The aim of this study was to investigate the possible underlying genetic and ecological explanations for these observations and the possibility of joint use of molecular markers and ecological parameters in the development of sustainable control strategies. A 28-month baseline monitoring and recovery activity was done in from 2004 in five regions in Kenya along an east-westerly transect. Monitoring and live sample collection was also done in the original outbreak area in eastern Kenya. There was greater LGB flight activity in western Kenya (high potential maize production area) than the low potential areas. Very few T. nigrescens were recovered, solely in the eastern regions. LGB flight activity followed a seasonal pattern mostly related to changes in the relative humidity at 12:00, rainfall and dew point temperature but with a 3 - 4 week lag. A linear predictive model based on these factors predicted 27 % of the observed flight activity. The survival and predation of five strains of T. nigrescens were compared at eight temperature levels between 15 °C and 36 °C at low and high humidity. All the strains of T. nigrescens exerted a significant reduction of LGB population build-up between 21 °C and 33 °C with generally better performance under humid conditions. There was no evidence of T. nigrescens development at 15 °C. At 18 °C, T. nigrescens oviposition and development was observed but the effect on LGB did not differ significantly from the control. The KARI population was the least effective in preventing grain damage at lower temperatures, but performed better than other strains above 30 °C at low humidity conditions. There was no control at 18 °C and 36 °C under both high and low humidity conditions. Since the extent of genetic differentiation in T. nigrescens was unclear from prior studies, several molecular marker techniques were progressively used. The RAPD-PCR did not reveal any genetic diversity between geographical populations. A 1000bp region of the mitochondrial mtCOI gene revealed two distinct clades differing consistently at 26 segregating sites. The two clades can be identified by simple PCR-RFLP procedure using single or double sequential restriction with EcoR1, HincII, RsaI and DdeI digestion. However, the two lineages co-exist among the mid-altitude Central American populations. The internal transcribed spacer regions ITS1 and ITS2 with some neighbouring coding sequences of the ribosomal DNA were cloned and sequenced. The spacer regions were so variable in length and sequence between T. nigrescens and related Histeridae species that direct sequence alignment was not meaningful. Within T. nigrescens, there was intragenomic variability of the spacer regions mostly involving insertions and deletions of variable tandem repeat units predominantly within the ITS regions. The short flanking coding (18S, 5.8S and 21S) regions were conserved across populations and six other Histeridae species. There was no significant secondary structure variation of the ITS regions among populations of T. nigrescens. Twenty-four novel variable microsatellite markers were developed and tested on the Honduras populations. Alleles per locus ranged between two and twelve with observed heterozygosity between 0.048 and 0.646. Six loci deviated significantly from Hardy Weinberg Equilibrium and possibly had null alleles. The success of microsatellite amplification in outgroup species and variability of markers declined with an increase in the phylogenetic distance between the test species and T. nigrescens. Genotyping 432 individuals from 13 geographic populations revealed a comparatively higher genetic diversity in field populations. Partial isolation by distance and time was observed. Population bottlenecks were not detected, but recent expansion was evident in laboratory populations. Although five dominant genetic clusters were identified by Bayesian methods, meaningful hierarchical population structure was observed at between two and nine population groups (p < 0.01; 10,000 iterations). Biological control of the larger grain borer using T. nigrescens seems an important aspect of the sustainable integrated control approach of the pest. Ecological adaptations, appropriate release strategies and genetic diversity are all essential considerations in these efforts and could be responsible for the variable success already observed. There is some genetic differentiation between populations of T. nigrescens but, further studies would be necessary to ascertain the contribution of such diversity to its predatory performance. The effect of laboratory culturing in aggravating genetic drift should be accommodated to avoid loss of diversity during sampling, quarantine, rearing and release of the predator. / Thesis (Ph.D. (Environmental Science))--North-West University, Potchefstroom Campus, 2009.

The larger grain borer (LGB) Prostephanus truncatus (Horn) is the most important pest of farm stored maize and cassava in Africa. This alien invasive species was introduced into the continent from Mesoamerica in the late 1970s and by 2008 had spread to at least 18 countries. In contrast to indigenous primary storage pests, LGB exists as on-farm and as wild populations, hence, sustainable control must target both environments. Biological control is especially attractive for wild populations to reduce early season grain store infestation, while cultural and chemical methods are useful to protect stored produce directly. Two populations of the predator Teretrius nigrescens Lewis were introduced into several African countries' as a biocontrol agent. It has shown long-term success and cost effective control in warm-humid areas. Control has however not been successful in cool and hot-dry zones. The aim of this study was to investigate the possible underlying genetic and ecological explanations for these observations and the possibility of joint use of molecular markers and ecological parameters in the development of sustainable control strategies. A 28-month baseline monitoring and recovery activity was done in from 2004 in five regions in Kenya along an east-westerly transect. Monitoring and live sample collection was also done in the original outbreak area in eastern Kenya. There was greater LGB flight activity in western Kenya (high potential maize production area) than the low potential areas. Very few T. nigrescens were recovered, solely in the eastern regions. LGB flight activity followed a seasonal pattern mostly related to changes in the relative humidity at 12:00, rainfall and dew point temperature but with a 3 - 4 week lag. A linear predictive model based on these factors predicted 27 % of the observed flight activity. The survival and predation of five strains of T. nigrescens were compared at eight temperature levels between 15 °C and 36 °C at low and high humidity. All the strains of T. nigrescens exerted a significant reduction of LGB population build-up between 21 °C and 33 °C with generally better performance under humid conditions. There was no evidence of T. nigrescens development at 15 °C. At 18 °C, T. nigrescens oviposition and development was observed but the effect on LGB did not differ significantly from the control. The KARI population was the least effective in preventing grain damage at lower temperatures, but performed better than other strains above 30 °C at low humidity conditions. There was no control at 18 °C and 36 °C under both high and low humidity conditions. Since the extent of genetic differentiation in T. nigrescens was unclear from prior studies, several molecular marker techniques were progressively used. The RAPD-PCR did not reveal any genetic diversity between geographical populations. A 1000bp region of the mitochondrial mtCOI gene revealed two distinct clades differing consistently at 26 segregating sites. The two clades can be identified by simple PCR-RFLP procedure using single or double sequential restriction with EcoR1, HincII, RsaI and DdeI digestion. However, the two lineages co-exist among the mid-altitude Central American populations. The internal transcribed spacer regions ITS1 and ITS2 with some neighbouring coding sequences of the ribosomal DNA were cloned and sequenced. The spacer regions were so variable in length and sequence between T. nigrescens and related Histeridae species that direct sequence alignment was not meaningful. Within T. nigrescens, there was intragenomic variability of the spacer regions mostly involving insertions and deletions of variable tandem repeat units predominantly within the ITS regions. The short flanking coding (18S, 5.8S and 21S) regions were conserved across populations and six other Histeridae species. There was no significant secondary structure variation of the ITS regions among populations of T. nigrescens. Twenty-four novel variable microsatellite markers were developed and tested on the Honduras populations. Alleles per locus ranged between two and twelve with observed heterozygosity between 0.048 and 0.646. Six loci deviated significantly from Hardy Weinberg Equilibrium and possibly had null alleles. The success of microsatellite amplification in outgroup species and variability of markers declined with an increase in the phylogenetic distance between the test species and T. nigrescens. Genotyping 432 individuals from 13 geographic populations revealed a comparatively higher genetic diversity in field populations. Partial isolation by distance and time was observed. Population bottlenecks were not detected, but recent expansion was evident in laboratory populations. Although five dominant genetic clusters were identified by Bayesian methods, meaningful hierarchical population structure was observed at between two and nine population groups (p < 0.01; 10,000 iterations). Biological control of the larger grain borer using T. nigrescens seems an important aspect of the sustainable integrated control approach of the pest. Ecological adaptations, appropriate release strategies and genetic diversity are all essential considerations in these efforts and could be responsible for the variable success already observed. There is some genetic differentiation between populations of T. nigrescens but, further studies would be necessary to ascertain the contribution of such diversity to its predatory performance. The effect of laboratory culturing in aggravating genetic drift should be accommodated to avoid loss of diversity during sampling, quarantine, rearing and release of the predator. / Thesis (Ph.D. (Environmental Science))--North-West University, Potchefstroom Campus, 2009.

O gênero Nocardia é composto por bactérias gram-positivas e filamentosas, que normalmente só causam doença em indivíduos imunocomprometidos. No entanto, certo número de infecções por nocardia têm sido relatados em pacientes imunocompetentes. Nos últimos anos, observou-se um aumento da frequência de infecções por Nocardia spp. e, com o aumento do número de espécies descritas, a identificação correta tem sido de difícil obtenção mas de grande importância para a aplicação do tratamento correto e elucidação epidemiológica. O objetivo deste estudo foi caracterizar, por métodos fenotípicos e moleculares, 72 isolados de Nocardia spp. de interesse médico, avaliando as metodologias para elaborar um algoritmo de identificação. Os isolados foram provenientes da Micoteca do Instituto de Medicina Tropical de São Paulo e da rotina do Núcleo de Tuberculose e Micobacterioses do Instituto Adolfo Lutz. Os isolados foram identificados por testes fenotípicos, identificação molecular por análise de restrição (PRA-hsp65), sequenciamento dos genes hsp65 e 16S rRNA e MALDI-TOF MS (Matrix-associated laser desorption time of flight mass spectrometry). O perfil de suscetibilidade foi analisado pelo método de Concentração Inibitória Mínima (MIC) e disco difusão (DD), com os fármacos: amicacina, ciprofloxacina, minociclina, tobramicina, amoxacilina+ácido clavulânico, imipenem e sulfametoxazol+trimetoprim. Os resultados revelaram que a identificação fenotípica foi insuficiente para definir as espécies. Apenas 24 (33,4%) isolados tiveram identificação fenotípica concordante com o sequenciamento do gene 16S rRNA. Na analise feita pela técnica de PRA-hsp65 foram observados 20 (27,8%) N. brasiliensis, seis (8,3%) isolados de outras espécies de nocardias e 38 (52,8%) foram considerados novos padrões (NP). Foi detectado um isolado misto e em cinco isolados não foi obtido produto de amplificação. O sequenciamento do gene hsp65 proporcionou a identificação de 51 isolados como Nocardia, 14 foram identificados como pertencentes a outros gêneros, dos quais, um apresentou mistura de nocardia e micobactéria, sendo identificado como Mycobacterium abscessus no gene hsp65 (análise in silico) e N. otitidiscaviarum no gene 16S rRNA. Sete isolados não foram sequenciados devido à ausência de amplificação do fragmento. Os isolados analisados através do sequenciamento do gene 16S rRNA, foram identificados como Nocardia (57-79,2%), Gordonia (7-9,7%), Rhodoccocus (3-4,2%), Tsukamurella (2-2,8%), Mycobacterium (2-2,8%) e Streptomyces (1-1,3%). Para a análise de MALDI-TOF MS foi observado que, dos 72 isolados estudados, 49 foram identificados como Nocardia, 11 como pertencentes a outros gêneros e 12 amostras não puderam ser identificadas devido aos valores de leitura não serem adequados para análise. Pela primeira vez o corante resazurina foi utilizado para leitura de MIC de Nocardia sp. Entre os fármacos testados através do MIC, os que apresentaram maior sensibilidade foram amicacina (100%) e tobramicina (84%). As maiores resistências foram encontradas com os fármacos sulfametoxazol+trimetoprim (76%) e imipenem (54%). Devido à ausência de critérios interpretativos de disco difusão para o gênero Nocardia, foi elaborado um critério para o presente estudo. Os resultados obtidos no teste de DD mostraram 100% de sensibilidade para os fármacos amicacina, minociclina e sulfametoxazol+trimetroprim. Os isolados apresentaram a maior porcentagem de resistência ao fármaco ciprofloxacina (64%). Comparando os resultados de DD com os obtidos no MIC, observamos que os fármacos ciprofloxacina, imipenem e sulfametoxazol+trimetoprim apresentaram porcentagem de discordância acima de 20%. O fármaco sulfametoxazol+trimetoprim teve a maior discordância (>75%), com elevada porcentagem de isolados resistentes na MIC, mas baixa porcentagem de resistência em DD. O único fármaco com 100% de concordância entre os resultados foi amicacina. Foi elaborado um algoritmo de identificação que utiliza técnicas fenotípicas para triagem para diferenciar nocardias de outros gêneros. A identificação por PRA-hsp65 será útil na rotina de laboratório de micobactérias como identificação presuntiva. A identificação definitiva das espécies deve ser obtida pelo sequenciamento do gene 16S rRNA. / The genus Nocardia comprises filamentous gram-positive bacteria that usually cause disease only in immunocompromised individuals. However, a number of Nocardia infections have been reported in immunocompetent patients. Overall, it has been reported in the recent years an increased frequency of infections caused by Nocardia spp. due to an also increasing number of species, making the correct identification of the species more difficult. Correct identification assumes a major importance with respect to antimicrobial treatment\'s choice as well a for epidemiological investigation purposes. The objective of this study was to characterize by phenotypic and molecular methods 72 isolates of Nocardia spp. of medical interest, designing the methodologies for an identification algorithm. The isolates were obtained from the fungal collection of the Tropical Medicine Institute of São Paulo and the routine service of the Tuberculosis and Mycobacteriosis Center of the Adolfo Lutz Institute. The isolates were identified by phenotypic testing, molecular identification by restriction analysis (PRA-hsp65), hsp65 and 16S rRNA genes sequencing, and MALDI-TOF MS (matrix-associated laser desorption time-of-flight mass spectrometry). The susceptibility profile was analyzed by the Minimum Inhibitory Concentration method (MIC) and disk diffusion (DD), with the following drugs: amikacin, ciprofloxacin, minocycline, tobramycin, amoxicillin+ clavulanic acid, imipenem and sulfamethoxazole trimethoprim. The results showed that the phenotypic identification was insufficient to define the species. Only 24 (33.4%) of the isolates had phenotype identification that was concordant with the 16S rRNA gene sequencing. In the analysis made with the PRA-hsp65 technique were observed 20 (27.8%) N. brasiliensis and six (8.3%) isolates from other species of Nocardia spp., while 38 isolates (52.8%) were considered as new standards (NP). Also by this technique a mixed isolate was detected and an amplification product was not obtained in five isolates. The hsp65 gene sequencing provided identification of 51 isolates as Nocardia, 14 were identified as belonging to other genera, one of them being identified either as Mycobacterium abscessus by in silico analysis of the hsp65 gene and as N. otitidiscaviarum by the 16S rRNA gene sequencing. Seven isolates were not sequenced due to the absence of the amplified fragment. The isolates analyzed by 16S rRNA gene sequencing were identified as Nocardia (57 to 79.2%), Gordonia (7 to 9.7%), Rhodoccocus (3 to 4.2%), Tsukamurella (2-2, 8%), Mycobacterium (2 to 2.8%) and Streptomyces (1-1.3%). By MALDI-TOF MS analysis of the 72 isolates, 49 were identified as Nocardia, 11 were identified as belonging to other genera and 12 isolates could not be identified because the samples provided reading values that were inadequate for analysis. For the first time, to our knowledge, the resazurin dye was used to determine the MICs of Nocardia sp. Among the drugs tested, the most sensitives were amikacin (100%) and tobramycin (84%). The higher resistances were found with trimethoprim-sulfamethoxazole (76%) and imipenem (54%). Due to the absence of establishe criteria for the interpretation of the disk diffusion assay with Nocardia, we designed a specific criterion for this study. The results obtained in the DD test showed 100% sensitivity for the drugs amikacin, minocycline and trimethoprim-sulfamethoxazole. The isolates showed the highest percentage of resistance to ciprofloxacin drug (64%). Comparing the results with those obtained with DD and MIC, we observed that ciprofloxacin, imipenem and sulfamethoxazole-trimethoprim showed a percentage of disagreement above 20%. The sulfamethoxazole-trimethoprim drug had the highest discrepancy (> 75%), with high percentage of resistant isolates with MIC but low percentage of resistance in DD. The only drug with 100% agreement between the both results was amikacin. We designed a recognition algorithm using phenotyping techniques to screen and differentiate nocardias from other genera. The identification by PRA-hsp65 will be useful in routine mycobacteria laboratory as a presumptive identification tool. The final identification of the species should be obtained by sequencing the 16S rRNA gene.

Orientador: Vera Lúcia Gil da Silva Lopes / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-19T02:52:06Z (GMT). No. of bitstreams: 1 Sgardioli_IlariaCristina_M.pdf: 3234786 bytes, checksum: cb2f0f67f9d7df7814742dbf6ed4fb44 (MD5) Previous issue date: 2011 / Resumo: A Síndrome Velocardiofacial (SVCF), uma das formas do espectro da Síndrome de deleção 22q11.2, possui incidência de 1/4.000 a 1/6.000 nascimentos. Embora a microdeleção em 22q11.2 seja a principal causa da síndrome, cerca de 10 a 20% dos pacientes com características clínicas da SVCF não a apresentam. Em alguns indivíduos com características clinicas da SVCF e sem microdeleção em 22q11.2, foram encontradas outras aberrações cromossômicas e mutações no gene TBX1. Existem evidências em modelos animais que aventam a associação de alterações no gene FGF8 ao fenótipo da SVCF em humanos. No entanto, até o momento, o gene FGF8 ainda não havia sido estudado em pacientes com SVCF sem microdeleção. Os objetivos deste trabalho foram: 1 - investigar as causas genéticas em pacientes com suspeita clínica da SVCF por meio de cariótipo e triagem de microdeleções, utilizando a técnica de MLPA e FISH; 2 - verificar a presença de alterações nas seqüências codificantes dos genes TBX1 e FGF8 em pacientes sem deleção 22q11.2. Foram incluídos 109 indivíduos com suspeita clínica de SVCF avaliados clinicamente por médico geneticista e os métodos utilizados foram: cariótipo com bandas G; MLPA, FISH; seqüenciamento direto das regiões codificantes dos genes TBX1 e FGF8 e SNP-array. Dos 101 casos em que o exame de cariótipo foi realizado, quatro apresentaram aberrações cromossômicas não relacionadas à SVCF, sendo que em duas foi possível a confirmação dos resultados por SNP-array. Realizou-se MLPA de 106 casos, sendo que 29 foram positivos para a deleção. Dos casos negativos, selecionou-se 31 indivíduos após reavaliação clínico-dismorfológica com a hipótese clínica mantida e foi realizado seqüenciamento das regiões codificantes dos genes TBX1 e FGF8. No gene TBX1 foram encontradas variações normais na seqüência e no gene FGF8 não foram detectadas alterações, com exceção dos exons 1 e 2, em que não foi possível análise por problemas técnicos. O exame de cariótipo contribuiu na investigação inicial e permitiu concluir a investigação por outras técnicas. O MLPA se mostrou eficiente para a investigação diagnóstica da deleção 22q11.2, identificando, também, outras alterações; FISH confirmou 82,1% dos resultados obtidos por MLPA. Não foram identificadas alterações patogênicas nas regiões codificantes dos genes TBX1 e em 90% das regiões codificantes do gene FGF8 / Abstract: Velocardiofacial Syndrome (SVCF), one of the forms of the 22q11.2 Microdeletion Syndrome spectrum, has an incidence of 1/4.000 to 1/6.000 births. Although the 22q11.2 microdeletion is the main cause of the syndrome, approximately 10 to 20% of the patients with clinical features of SVCF don't present it. In a few individuals with SVCF clinical features and without 22q11.2 microdeletion other chromosomal aberrations and changes in TBX1 gene were found. There is evidence in animal models that suggests the associated changes in FGF8 gene in humans. However, the FGF8 gene had not been studied in patients with SVCF without microdeletion yet. The purpose of this work was: 1 - To investigate the genetic causes in patients with clinical suspicion of SVCF through the karyotype and microdeletion screening using MLPA and FISH techniques; 2 - To check the presence of changes in coding sequences of the TBX1 gene and FGF8 gene in patients without 22q11.2 deletion. 109 individuals with clinical suspicion of SVCF and clinically evaluated by a clinical geneticist were included, and the following methods were used: karyotype with GTG banding, MLPA, FISH, direct sequencing of coding regions of TBX1 and FGF8 gene and SNP-array. Four out of 102 cases in which the tests of karyotype were performed presented chromosomal aberrations not related to SVCF, two of them confirmed by SNP-array. The MLPA of 106 cases was performed, 29 of them positive to deletion. 31 individuals were selected among negative cases, after clinical reevaluation based on the same clinical hypothesis maintained, and direct sequencing of coding regions of TBX1 and FGF8 gene were performed. Normal variations in sequence were found in TBX1 gene and alterations were not detected in FGF8 gene except for 1 and 2 exons because of technical problems. The karyotype test contributed in the first investigation and permitted us to conclude the investigation by means of other techniques. The MLPA proved to be effective for the diagnostic investigation of 22q11.2 deletion, identifying other alterations as well; FISH confirmed 82,1% results obtained by MLPA. Pathogenic alterations in coding regions of TBX1 gene and in 90% of the coding regions of FGF8 gene were not identified / Mestrado / Ciencias Biomedicas / Mestre em Ciências Médicas

Abstract The mitochondrial oxidative phosphorylation system (OXPHOS) generates energy but also deleterious reactive oxygen species (ROS). Changes in the cytoskeleton, composed mainly of microfilaments, microtubules and intermediate filaments, have been observed in OXPHOS deficiency. The 3243A>G point mutation in mitochondrial DNA (mtDNA) leads to mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS), which is the most common mitochondrial disease. Interestingly, mitochondrial aberrations have been demonstrated in patients with a mutation in NOTCH3, the genetic cause of cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). Randomization of vimentin intermediate filament direction and length together with slower population growth was observed in myoblasts with 3243A>G, with no difference in the amount of apoptotic cell death. Upon complex IV inhibition (with or without the microtubule-depolymerizing compound nocodazole) or a lack of mtDNA (ρ0) in osteosarcoma cells the vimentin network collapsed perinuclearly, forming thick bundles, whereas complex I inhibition led to thinner vimentin network bundles. Furthermore, the amount of vimentin was increased in ρ0 cells. Mitochondria accumulated around the nucleus upon complex IV inhibition and in ρ0 cells. Analysis of the total proteome revealed that specific OXPHOS deficiencies led to changes in the expression of cytoskeletal proteins and proteins involved in apoptosis, OXPHOS, glycolysis and oxidative stress response. Muscle histochemical and genetic analysis showed ragged red fibres and cytochrome c oxidase-negative fibres to be associated with 5650G>A in a patient with R133C in NOTCH3 and 5650G>A in MTTA. Immunolabelling of cells with R133C and 5650G>A revealed a sparse tubulin network with asters and less abundant mitochondria by comparison with control cell lines. Comparison of nucleotide diversity between CADASIL pedigrees and controls showed increased mtDNA sequence variation in the CADASIL patients. Also maternal relatives in two CADASIL pedigrees differed from each other in their mtDNA. These findings suggest that defects in OXPHOS lead to selective changes in the vimentin network, which may have a role in the pathophysiology of mitochondrial diseases. They also suggest a relationship between NOTCH3 and mtDNA, and establish the pathogenicity of 5650G>A. The overall results emphasize that a deficiency in the energy converting system together with oxidative stress can lead to cytoskeletal changes.

ABSTRACT COMPARATIVE SEQUENCE ANALYSIS OF THE INTERNAL TRANSCRIBED SPACER 2 REGION OF TURKISH RED PINE (Pinus brutia TEN.) AND NATURAL ALEPPO PINE (Pinus halepensis MILL.) POPULATIONS FROM TURKEY Tozkar, &Ouml / zge M.S., Department of Biology Supervisor: Prof. Dr. Zeki Kaya April, 2007, 107 pages Turkish red pine (Pinus brutia) is wide-spread and an important forest tree species in Turkey, occurring mainly in southern, western and north-western Turkey and as small isolated populations in the Black Sea region. Aleppo pine (Pinus halepensis) has naturally found only in Adana and Mugla provinces as small population in mixture with Turkish red pine. Although Turkish red pine and Aleppo pine are morphologically different, Turkish red pine has been regarded as subspecies of Aleppo pine by some taxonomists due to occurrence of natural hybridization between these two species. However, the phylogenic relationship between these species needs to be explored further. In the present study, by sampling overlapped populations of both species from Mugla and Adana provinces (4 populations of Turkish red pine and 3 populations of Aleppo pine), internal transcribed spacer (ITS) region of ribosomal DNA were comparatively studied with sequence analysis. Although ITS1, 5.8s and ITS2 regions of ribosomal DNA were studied with ITS primers, only ITS2 region was successfully amplified with polymerase chain reaction (PCR). The complete data set for this region was analysed using MEGA3.1 and Arlequin softwares. Analysis of molecular variance (AMOVA) demonstrated the highest genetic differentiation between Turkish red pine and Aleppo pine in Mugla with 100 percentage of variation. AMOVA analysis also indicated the possibility of low-level migration of genes between Turkish red pine and Aleppo pine populations in Adana with 50.65 percent of molecular variance. Haplotype comparison revealed that two major haplotypes were represented Based on the results of ITS2 region sequence analysis, Turkish populations of Aleppo pine and Turkish red pine populations could not be fully differentiated. In Mugla province Turkish red pine and Aleppo pine revealed more differentiation due to reproductive isolation. But in Adana province, two species shared more common genetic background due to possible hybridization. Since ITS2 region of nuclear ribosomal DNA revealed a few variable and parsimony informative sites for both species, thus, only ITS2 region of ribosomal DNA does not appear to be sufficient for fully resolving genetic relationships between Turkish red pine and Aleppo pine populations. Further studies including ITS1 and 5.8s regions of ribosomal DNA and populations included from major Aleppo pine distribution areas will be useful to understand the evolutionary relationship between Aleppo pine and Turkish red pine populations in Turkey.

With recent and continuing advances in bioinformatics, the volume of sequence data has increased tremendously. Along with this increase, there is a growing need to develop efficient algorithms to process such data in order to make useful and important discoveries. Careful analysis of genomic data will benefit science and society in numerous ways, including the understanding of protein sequence functions, early detection of diseases, and finding evolutionary relationships that exist among various organisms. Most sequence analysis problems arising from computational genomics and evolutionary biology fall into the class of NP-complete problems. Advances in exact and approximate algorithms to address these problems are critical. In this thesis, we investigate a novel graph theoretical model that deals with fundamental evolutionary problems. The model allows incorporation of the evolutionary operations ``insertion', ``deletion', and ``substitution', and various parameters such as relative distances and weights. By varying appropriate parameters and weights within the model, several important combinatorial problems can be represented, including the weighted supersequence, weighted superstring, and weighted longest common sequence problems. Consequently, our model provides a general computational framework for solving a wide variety of important and difficult biological sequencing problems, including the multiple sequence alignment problem, and the problem of finding an evolutionary ancestor of multiple sequences. In this thesis, we develop large scale combinatorial optimization techniques to solve our graph theoretical model. In particular, we formulate the problem as two distinct but related models: constrained network flow problem and weighted node packing problem. The integer programming models are solved in a branch and bound setting using simultaneous column and row generation. The methodology developed will also be useful to solve large scale integer programming problems arising in other areas such as transportation and logistics.

Orientadores: Reginaldo Palazzo Junior, Marcio de Castro Silva Filho / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia Eletrica e de Computação / Made available in DSpace on 2018-08-15T16:43:49Z (GMT). No. of bitstreams: 1 Rocha_AndreaSantosLeiteda_D.pdf: 5117477 bytes, checksum: e8f0c742c67382cad01387d3e62f6705 (MD5) Previous issue date: 2010 / Resumo: Um dos desafios em biologia matemática e mostrar a existência de qualquer forma de códigos corretores de erros na estrutura do DNA. Usando os conceitos da teoria de comunicação, propomos um modelo para o sistema de codificacao e decodificaçao do mecanismo de importaçao de proteínas mitocondriais similar a um sistema de comunicacoes digital. Este modelo consiste de um mapeador responsável por transformar os nucleotídeos (A, C, G, T) no alfabeto (0,1, 2, 3) usado pelo codigo sobre a estrutura de anel; um codificador (cádigo BCH); e um modulador (codigo genetico, tRNA e rRNA). O processo de decodificaçao baseia-se em uma analogia entre o processo de decodificacão do algoritmo Berlekamp-Massey para aneis e o complexo TOM (complexo ancorado na membrana externa da mitocondria responsavel por auxiliar na importacçãao das proteínas precursoras). Neste processo temos um demodulador (proteínas Tom 70 e Tom20), um decodificador (o complexo GIP - poro geral de inserção) e o receptor (subcompartimento mitocondrial). Neste trabalho mostramos que as sequencias de DNA (sequencias de direcionamento) são identificadas como palavras-codigo de um código G-linear sobre a extensão de um anel de Galois. Além disso, essas sequências de DNA e suas fitas complementares estão relacionadas matematicamente através dos polinómios primitivos e seus polinómios recíprocos, respectivamente. Um estudo filogenético sugere que a proteína malato desidrogenase da Arabidopsis thaliana encontrada no banco de dados NCBI e uma sequência derivada da proteína malato desidrogenase reproduzida pelo cídigo corretor de erros. Este modelo também reproduz com notível precisão os parâmetros cinéticos baseados em substituicões de aminoíacidos em oligopeptídeos sintéticos. Apresentamos, pela primeira vez, a existência de códigos corretores de erros associados com as sequências de DNA, os quais sugerem fortemente a existência de códigos concatenados no genoma. Os resultados apresentados neste trabalho contribuem para o desenvolvimento de um procedimento sistemático que podera ser empregado em analises de mutacães/polimorfismos com aplicações na engenharia geníetica. / Abstract: One of the puzzling problems in mathematical biology is to show the existence of any form of error-correcting code in the DNA structure. Using information theory considerations we propose a model for the biological coding system similar to that of a digital communication system. This model consists of a mapper (transformations from the set of nucleotides either to the set (0, 1, 2, 3) ring; an encoder (BCH code); and a modulator (genetic code, tRNA and rRNA). The decoding process is based on the Modified Berlekamp-Massey algothm in an analogy with the TOM complex (translocase of the mitochondrial outer membrane). In this process we have a demodulator (Tom 70 and Tom 20 proteins), a decoder (GIP complex) and the receiver (mitochondrion). In this work we show that DNA sequences (targeting sequences) are identified as codewords of a G-linear code over Galois ring extensions. In addition, these DNA sequences and their complementary strands are mathematically related to the primitive polynomials and their reciprocal polynomials, respectively. A phylogenetic study suggest that the MDH protein, Arabidopsis thaliana, found in the NCBI databank is a derived sequence of the MDH protein reproduced by the error correcting code. This model also reproduces with remarkable accuracy kinetic parameters based on amino acid substitutions on synthetic oligopeptides. We show, for the first time, the existence of error-correcting codes associated with DNA sequences, which strongly infer on the existence of nested codes within the genome. The results presented in this work contribute to the development of a systematic procedure which may be employed in the mutations/polymorphisms analysis with applications in genetic engineering. / Doutorado / Telecomunicações e Telemática / Doutor em Engenharia Elétrica

Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-06-30T20:05:29Z No. of bitstreams: 1 thaisaparecidavieirareis.pdf: 1630302 bytes, checksum: d9e1d09066119dbc3c4a339f7ccc6564 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2016-07-13T15:59:16Z (GMT) No. of bitstreams: 1 thaisaparecidavieirareis.pdf: 1630302 bytes, checksum: d9e1d09066119dbc3c4a339f7ccc6564 (MD5) / Made available in DSpace on 2016-07-13T15:59:16Z (GMT). No. of bitstreams: 1 thaisaparecidavieirareis.pdf: 1630302 bytes, checksum: d9e1d09066119dbc3c4a339f7ccc6564 (MD5) Previous issue date: 2012-06-29 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais / A doença diarréica aguda (DDA) é, ainda hoje, uma das principais causas de morbidade e mortalidade infantil, nos países em desenvolvimento. Na diarreia aguda não bacteriana, os adenovírus entéricos constituem um dos importantes agentes etiológicos da doença. Os adenovírus humanos (HAdV) pertencem à família Adenoviridae e ao gênero Mastadenovirus, cujos membros estão classificados em 7 espécies (A-G) e 54 sorotipos. Dentre estes, os sorotipos 40 e 41, ambos da espécie F, são os mais comumente associados com a DDA. Considerando-se, então, a importância da DDA em países em desenvolvimento, o grande número de casos que não são esclarecidos e o pouco conhecimento sobre a infecção e a participação dos HAdV na gênese da DDA, em Juiz de Fora, Minas Gerais, foi realizado o presente estudo. Entre janeiro de 2007 e dezembro de 2010, foram analisados 395 espécimes fecais diarréicos, provenientes de indivíduos de várias idades, atendidos em serviços ambulatoriais e hospitalizados. A presença dos HAdV foi detectada pela reação de PCR , utilizando-se os iniciadores específicos e a caracterização molecular das amostras positivas foi feita pelo seqüenciamento e análise filogenética das sequências parciais do gene do Hexon. Para as análises estatísticas, foi utilizado o programa SPSS versão 13.0, tendo se adotado um valor de significância como p<0,05. A prevalência da infecção por HAdV, no período 2007-2010, foi de 10,9% (43/395). Os resultados mostraram que não houve correlação significante entre a procedência da amostra (ambulatorial X hospitalar) e a ocorrência da infecção (p=0,152), o mesmo tendo sido observado em relação ao gênero do indivíduo infectado (p=0,393). Por outro lado, a maioria dos casos positivos foi detectada em crianças de até 24 meses de idade, mostrando uma correlação estatisticamente significante entre a idade dos indivíduos infectados e a ocorrência da infecção (p=0,007). Na maioria dos casos de infecção pelo HAdV (36/43), este foi o único agente viral detectado, no entanto, foram observados casos de coinfecção HAdV/Rotavirus (5/43) e HAdV/Norovirus (2/43). A análise filogenética das sequências parciais do gene do Hexon, de 35 amostras positivas, revelou que todas agruparam com amostras de HAdV da espécie F, sorotipo 41, confirmando assim, a associação de HAdV entéricos, nos casos estudados. Este levantamento epidemiológico revelou a presença e a circulação destes vírus na população de Juiz de Fora, no período avaliado, bem como sua importante participação na gênese da DDA, permitindo assim, esclarecer uma boa parte dos casos da doença, que normalmente ficaria sem definição etiológica. / Acute diarrheal disease (ADD) is still the major cause of child morbidity and mortality in developing countries. Among the non-bacterial diarrhea, enteric adenoviruses are one of the most important etiologic agents of disease. Human adenoviruses (HAdV) belongs to the Adenoviridae family and Mastadenovirus genus. The virus are classified into seven species (A-G) and 54 serotypes. Among them, serotypes 40 and 41, both of the species F, are the most commonly associated with ADD. Taking in consideration the importance of the DDA in developing countries, the large number of cases that don’t have the etiologic agent identified and the lack of knowledge about the participation of HAdV infection and the pathogenesis of ADD, we performed this study in Juiz de Fora, Minas Gerais. Between January of 2007 and December of 2010 395 diarrheal fecal specimens originating from individuals of various ages treated in ambulatory and hospitalized were analyzed. The presence of HAdV was detected by PCR, using specific primers, and molecular characterization of positive samples was performed by sequencing and phylogenetic analysis of partial sequences of the hexon gene. For statistical analyzes, we used SPSS version 13.0, adopting a value of p <0.05 as significant. The prevalence of infection by HAdV between 2007-2010 was 10.9% (43/395). The results showed no significant correlation between the origin of the sample (hospital X ambulatory) and the occurrence of infection (p = 0.152), and the same was observed in relation to gender of the infected person (P=0,393). Moreover, the majority of positive cases was detected in children under 24 months of age, showing a statistically significant correlation between the age of the infected individuals and the occurrence of infection (p=0,007). In most cases of infection HAdV (36/43), this was the only viral agent detected, however, cases of co-infection HAdV / Rotavirus (5/43) and HAdV /Norovirus (2/43) were identified. Phylogenetic analysis of partial sequences of the hexon gene from 35 positive samples revealed that all samples clustered with HAdV species F, serotypes 41, confirming the association of enteric HAdV in the cases of this study. This epidemiological survey revealed the presence and circulation of these viruses in the population of Juiz de Fora in the period studied, as well as its important role in the genesis of the DDA, our data identified a good number of cases of the disease, which normally remains unidentified.