Diss. (sammanfattning) Göteborg : Univ. , 2009. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Texas at Austin, 2007. / Vita. Includes bibliographical references.

Master of Science / Agronomy / Mithila Jugulam / Common waterhemp (Amaranthus rudis) is a problematic weed species of cropping systems throughout the Midwestern states, including Kansas. Recently, waterhemp populations from Kansas were found to have evolved resistance to the widely used herbicide glyphosate as a result of amplification of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), the enzyme target of glyphosate. The objectives of this research were to 1) perform glyphosate dose-response study and determine the relationship between relative EPSPS genomic copies and EPSPS gene expression in glyphosate-resistant waterhemp, and 2) characterize the genomic configuration and distribution of EPSPS copies using florescence in situ hybridization (FISH) in three glyphosate-resistant waterhemp populations. Waterhemp populations from eastern Kansas were screened with 868 g ae haˉ¹ (field used rate) of glyphosate, and genomic DNA and total RNA was isolated from the survivors to determine the EPSPS genomic copies and EPSPS gene expression relative to the acetolactate synthase (ALS) gene using qPCR. Furthermore, waterhemp specific EPSPS probes were synthesized to perform florescence in situ hybridization (FISH) on these glyphosate-resistant plants. Results of these experiments indicate a positive correlation between level of glyphosate resistance, EPSPS copies, and their expression. As expected, a negative correlation was found between shikimate accumulation and EPSPS copies. Sequencing of the EPSPS gene showed no presence of the proline 106 mutation, which is known to be associated with glyphosate resistance suggesting that an insensitive EPSPS enzyme was not involved in the mechanism of glyphosate resistance. FISH analysis of resistant plants illustrated presence of amplified EPSPS copies on two homologous chromosomes, likely near the centromeric region. . This is the first report demonstrating a positive relationship between EPSPS copies and expressions, as well as chromosome configuration of EPSPS copies in glyphosate- resistant waterhemp from Kansas.

Background The lack of an effective diagnostic tool for Carrion’s disease leads to misdiagnosis, wrong treatments and perpetuation of asymptomatic carriers living in endemic areas. Conventional PCR approaches have been reported as a diagnostic technique. However, the detection limit of these techniques is not clear as well as if its usefulness in low bacteriemia cases. The aim of this study was to evaluate the detection limit of 3 PCR approaches. Methodology/Principal Findings We determined the detection limit of 3 different PCR approaches: Bartonella-specific 16S rRNA, fla and its genes. We also evaluated the viability of dry blood spots to be used as a sample transport system. Our results show that 16S rRNA PCR is the approach with a lowest detection limit, 5 CFU/μL, and thus, the best diagnostic PCR tool studied. Dry blood spots diminish the sensitivity of the assay. Methodology/Principal Findings We determined the detection limit of 3 different PCR approaches: Bartonella-specific 16S rRNA, fla and its genes. We also evaluated the viability of dry blood spots to be used as a sample transport system. Our results show that 16S rRNA PCR is the approach with a lowest detection limit, 5 CFU/μL, and thus, the best diagnostic PCR tool studied. Dry blood spots diminish the sensitivity of the assay. Conclusions/Significance From the tested PCRs, the 16S rRNA PCR-approach is the best to be used in the direct blood detection of acute cases of Carrion’s disease. However its use in samples from dry blood spots results in easier management of transport samples in rural areas, a slight decrease in the sensitivity was observed. The usefulness to detect by PCR the presence of low-bacteriemic or asymptomatic carriers is doubtful, showing the need to search for new more sensible techniques.

Orientador: Gustavo Vitti Môro / Banca: Rinaldo Cesar de Paula / Banca: Bruno Henrique Sardinha de Souza / Resumo: Os híbridos de milho transgênicos, em geral, apresentam o locus transgênico em hemizigose. O objetivo do trabalho foi avaliar o efeito do número de alelos transgênicos em híbridos de milho em relação à expressão de Cry1F nas folhas, ataque de Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) e a produtividade de grãos, utilizando cinco híbridos isogênicos nas versões transgênicas homozigota e hemizigota, além da versão convencional de um dos híbridos. O nível de expressão da proteína Cry1F (PRYF) foi quantificado pela técnica de ELISA. Nos experimentos de campo, conduzidos na primeira e segunda safras do ano agrícola 2015/2016, avaliou-se o ataque de S. frugiperda em campo (ALC), por infestação natural, e a produtividade de grãos (PG). Dois bioensaios foram realizados em laboratório para avaliar a sobrevivência larval de S. frugiperda de 1º instar (SL) alimentadas com as folhas dos híbridos. Os híbridos transgênicos, homozigotos e hemizigotos, não apresentaram silenciamento gênico. Os híbridos homozigotos apresentaram maior concentração de proteína Cry1F. Quando houve elevado ALC, na primeira safra, os híbridos transgênicos foram superiores à testemunha convencional na PG, entretanto, não houve diferença entre os híbridos homozigotos e hemizigotos. Os híbridos transgênicos também foram superiores à testemunha convencional nos bioensaios, sendo que os homozigotos apresentaram as menores médias de SL. A presença de um alelo transgênico a mais nos híbridos homozigotos ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstracts: Genetically modified (GM) maize hybrids, in general, possess the transgenic locus in a hemizygous state. The aim of the study was to assess the effect of the number of transgenic alleles in maize hybrids regarding the Cry1F leaf expression, Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) attack and grain yield, through the evaluation of five isogenic hybrids in the homozygous and hemizygous transgenic versions and a non-GM hybrid. Cry1F protein expression levels (PRYF) were quantified by ELISA. In field experiments conducted during the summer and autumn seasons of 2015/2016, we assessed the leaf-feeding injury of S. frugiperda in the field (LFI) by natural infestation and grain yield (GY). Two bioassays were carried out in the laboratory to evaluate the survival of first-instar S. frugiperda larvae (SL) fed with the maize hybrids. Transgenic hybrids did not present gene silencing. Homozygous hybrids presented higher Cry1F expression levels than hemizygous hybrids. With high LFI during the summer season, transgenic hybrids were superior to the non-GM for GY, however, there was no difference between homozygous and hemizygous hybrids. The transgenic hybrids were superior to the non-GM hybrid in the bioassays, and the homozygotes caused the highest mortality of S. frugiperda. The addition of one transgenic allele in the homozygous hybrids provided an additive genetic effect, increasing PRYF and S. frugiperda control, whereas GY was not affected. In conclusion, there are no limitations to the use of transgenic homozygous maize hybrids, which presented better S. frugiperda control comparing to hemizygous hybrid. / Mestre

A planta daninha Amaranthus palmeri é nativa dos Estados Unidos, porém foi pela primeira vez relatada no Brasil no ano de 2015. Embora comprovadamente com resistência múltipla aos herbicidas inibidores da ALS e da EPSPS, até o momento não foram investigadas as bases moleculares da resistência. Além disso, por causa da recente introdução da planta daninha no país, alternativas de manejo com culturas tolerantes a herbicidas necessitam ser estudadas. Sendo assim, os objetivos desse trabalho são de caracterizar a espécie de planta daninha introduzida no país, identificar os mecanismos de resistência aos herbicidas inibidores da ALS e da EPSPS presentes no biótipo, e propor abordagens de manejo em ambientes dos novos eventos transgênicos resistentes a herbicidas. Um bioensaio utilizando marcadores genéticos foi desenvolvido para confirmar que a população coletada no estado do Mato Grosso (BR-R) é A. palmeri, e não A. tuberculatus, outra espécie dióica do gênero Amaranthus. Os resultados de experimentos de curvas de dose-resposta e acúmulo de chiquimato indicaram que a BR-R possui alto nível de resistência, com DL50 de 4.426 e 3.400 g glyphosate ha-1 no primeiro e segundo experimento, respectivamente, mais que o dobro da dose típicamente recomendada para o controle da espécie e, adicionalmente, observou se acúmulo mínimo de chiquimato a concentração de 1 mM nos tecidos das plantas tratadas com o herbicida. BR-R também foi resistente a herbicidas dos grupos químicos das sulfoniluréias e imidazolinonas. O mecanismo de resistência ao glyphosate encontrado nesta população foi a super expressão gência, através do aumento no número de cópias do gene da EPSPS no genoma da planta BR-R, entre 50 e 179 cópias adicionais. Além disso, duas substituições de aminoácidos foram observadas na sequência da ALS, W574L e S653N, conferindo resistência tanto a sulfoniluréias quanto a imidazolinonas. No experimento utilizandos os herbicidas correspondentes às culturas geneticamente modificadas com novos traits de tolerância a herbicidas observou se, de uma forma geral, que as associações de herbicidas apresentaram níveis de controle mais satisfatórios. Assim, esta pesquisa confirma a introdução de da espécie A. palmeri no Brasil, assim como a resistência múltipla aos herbicidas inibidores da EPSPS e da ALS. Seu manejo é mais eficaz através da associação de herbicidas, garantindo assim o uso racional das novas tecnologias de culturas geneticamente modificadas com tolerância a herbicidas. / Palmer Amaranth (Amaranthus palmeri) is a weed species native to the United States, but it was reported in Brazil for the first time in 2015. Despite this population being resistant to EPSPS and ALS inhibitors, the molecular basis of its multiple resistance is unknown up to date. Because of this species introduction to Brazil, alternatives of management with the new herbicide-tolerant crops technologies need to be studied. The objectives of this research are to characterize the weed species introduced to Brazil, identify the mechanisms conferring resistance to ALS and EPSPS inhibitors herbicides, and to propose management approaches in environments with the new genetically modified herbicide-tolerant crops. A genotyping bioassay using genetic markers was developed to confirm that the species collected in the state of Mato Grosso (BR-R) is indeed A. palmeri and not A. tuberculatus, another dioceous species in the Amaranthus genus. Dose-response experiments and shikimate accumulation bioassay data indicate high level of resistance, with LD50 of 4,426 and 3,400 g glyphosate ha-1 in the first and second experiments, respectively, higher than the double rate tipically recommended to control it, and minimal accumulation in BR-R with 1 mM of glyphosate in treated plants in the leaf disks assay. BR-R also was resistanto to sulfonilurea and imidazolinone herbicides. The mechanism conferring resistance to glyphosate identified in this population was gene amplification, with increased EPSPS copy number - between 50 and 179 more copies in BR-R. Besides, two target-site mutations were identified in the ALS gene sequencing, W574L and S653N, conferring resistance to sulfonilureas and imidazolinones. The weed control experiment, overal, herbicide tank mixtures achieved higher levels of control. Therefore, this research confirms the introduction of A. palmeri to Brazil, as well as its multiple resistance to EPSPS and ALS inhibitor herbicides. Its control is more efficient with herbicide mixtures, which guarantees more susteinable use of the new herbicide-tolerant crop technologies.

A amplificação gênica pode ser compreendida como um mecanismo de regulação de expressão protéica em Leishmania. A exposição a concentrações não letais de diferentes drogas isoladamente, como o metotrexato, a primaquina, cloroquina e antimônio levam à amplificação de regiões específicas como as localizadas no cromossomo 6 e no cromossomo 23. Essas linhagens tornam-se resistentes a estas drogas e eventualmente apresentam resistência cruzada a outras. A terbinafina é uma substância sintética utilizada como antifúngico que age pela inibição da esqualeno epoxidase impedindo a síntese de ergosterol, componente da membrana celular de Leishmania. A exposição do parasita à terbinafina leva à amplificação da região H. A fragmentação da região H permitiu delimitar a resistência a 2,8 kb; fragmento T1. Por mutagênese insercional o gene de resistência à terbinafina foi definido e nomeado HTBF. A proteína codificada pelo HTBF possui uma extremidade N-terminal hidrofílica e C-terminal hidrofóbica com quatro alfa-hélices sendo, supostamente, uma proteína integral de membrana. Possui semelhança com a proteína Yip de Saccharomyces cerevisiae, que participa do transporte vesicular do retículo endoplasmático para o complexo de Golgi. O gene HTBF foi detectado em linhagem sensível de L. major e de outras espécies de Leishmania e seu transcrito em linhagens sensíveis e resistentes de L. major. Levantou-se a hipótese de se tratar de locus de resistência a múltiplas drogas já que as regiões amplificadas, mesmo que induzidas por uma determinada droga, podem levar a resistência a outras. A co-transfecção do gene de resistência ao antimonial MRPA no transfectante portador do HTBF levou à diminuição em até cinco vezes da capacidade de resistência ao antimônio. Apesar dos genes de resistência amplificarem juntos na região H, foi constatado que possuem ação contrária. Sendo que a terbinafina interfere na integridade da membrana celular, levantou-se hipótese na qual a resistência envolveria o mecanismo de reparo de membrana. A perda do fenótipo de resistência pela falta de cálcio sugerindo a inativação desse mecanismo é um fenômeno previsto corroborando a hipótese. / Gene amplification can be recognized as a protein regulation mechanism in Leishmania. Exposure to non-lethal concentrations of different drugs in isolated manner, such as, methotrexate, primaquine, chloroquine and antimony drives the amplification of specific regions. Examples for amplified regions are contained in chromosomes six and 23. The strains become resistant to these drugs and eventu-ally to other drugs as well. Terbinafine is a synthetic substance used as an antifungal agent. Its mechanism of action is by squalene epoxidase inhibition, blocking ergosterol synthesis, a cell membrane component in Leishmania. Parasite exposure to terbinafine elicits H region amplification. H region fragmentation allowed resistance delimitation to 2.8kb, T1 fragment. By insertional mutagenesis terbinafine resistance gene was defined and termed HTBF. HTBF coded protein has a hydrophilic N-terminal and a hydrophobic C-terminal containing four alpha-helixes, putatively, an integral membrane protein. It possesses homology to Saccharomyces cerevisiae Yip protein, which takes part in the vesicular transport from the endoplasmic reticulum to Golgi apparatus. HTBF gene was detected in sensitive L. major strains and other Leishmania species and its transcript was detected in both resistant and sensitive L. major strains. We raised the hypothesis of a multiple drug resistance locus, since amplified regions induced by one drug could elicit resistance to a second drug. Co-transfection of MRPA to the HTBF transfectant led to a five-fold decrease to antimonial resistance level. Even though these resistance genes amplify together in the H region, they have antagonizing mechanisms of action. Since terbinafine action disrupts membrane integrity, we raised a hypothesis in which resistance involves enhancement of membrane repair machinery. Loss of phenotype caused by the lack of calcium suggests the inactivation of this machinery is a predicted phenomenon, corroborating the hypothesis.

A planta daninha Amaranthus palmeri é nativa dos Estados Unidos, porém foi pela primeira vez relatada no Brasil no ano de 2015. Embora comprovadamente com resistência múltipla aos herbicidas inibidores da ALS e da EPSPS, até o momento não foram investigadas as bases moleculares da resistência. Além disso, por causa da recente introdução da planta daninha no país, alternativas de manejo com culturas tolerantes a herbicidas necessitam ser estudadas. Sendo assim, os objetivos desse trabalho são de caracterizar a espécie de planta daninha introduzida no país, identificar os mecanismos de resistência aos herbicidas inibidores da ALS e da EPSPS presentes no biótipo, e propor abordagens de manejo em ambientes dos novos eventos transgênicos resistentes a herbicidas. Um bioensaio utilizando marcadores genéticos foi desenvolvido para confirmar que a população coletada no estado do Mato Grosso (BR-R) é A. palmeri, e não A. tuberculatus, outra espécie dióica do gênero Amaranthus. Os resultados de experimentos de curvas de dose-resposta e acúmulo de chiquimato indicaram que a BR-R possui alto nível de resistência, com DL50 de 4.426 e 3.400 g glyphosate ha-1 no primeiro e segundo experimento, respectivamente, mais que o dobro da dose típicamente recomendada para o controle da espécie e, adicionalmente, observou se acúmulo mínimo de chiquimato a concentração de 1 mM nos tecidos das plantas tratadas com o herbicida. BR-R também foi resistente a herbicidas dos grupos químicos das sulfoniluréias e imidazolinonas. O mecanismo de resistência ao glyphosate encontrado nesta população foi a super expressão gência, através do aumento no número de cópias do gene da EPSPS no genoma da planta BR-R, entre 50 e 179 cópias adicionais. Além disso, duas substituições de aminoácidos foram observadas na sequência da ALS, W574L e S653N, conferindo resistência tanto a sulfoniluréias quanto a imidazolinonas. No experimento utilizandos os herbicidas correspondentes às culturas geneticamente modificadas com novos traits de tolerância a herbicidas observou se, de uma forma geral, que as associações de herbicidas apresentaram níveis de controle mais satisfatórios. Assim, esta pesquisa confirma a introdução de da espécie A. palmeri no Brasil, assim como a resistência múltipla aos herbicidas inibidores da EPSPS e da ALS. Seu manejo é mais eficaz através da associação de herbicidas, garantindo assim o uso racional das novas tecnologias de culturas geneticamente modificadas com tolerância a herbicidas. / Palmer Amaranth (Amaranthus palmeri) is a weed species native to the United States, but it was reported in Brazil for the first time in 2015. Despite this population being resistant to EPSPS and ALS inhibitors, the molecular basis of its multiple resistance is unknown up to date. Because of this species introduction to Brazil, alternatives of management with the new herbicide-tolerant crops technologies need to be studied. The objectives of this research are to characterize the weed species introduced to Brazil, identify the mechanisms conferring resistance to ALS and EPSPS inhibitors herbicides, and to propose management approaches in environments with the new genetically modified herbicide-tolerant crops. A genotyping bioassay using genetic markers was developed to confirm that the species collected in the state of Mato Grosso (BR-R) is indeed A. palmeri and not A. tuberculatus, another dioceous species in the Amaranthus genus. Dose-response experiments and shikimate accumulation bioassay data indicate high level of resistance, with LD50 of 4,426 and 3,400 g glyphosate ha-1 in the first and second experiments, respectively, higher than the double rate tipically recommended to control it, and minimal accumulation in BR-R with 1 mM of glyphosate in treated plants in the leaf disks assay. BR-R also was resistanto to sulfonilurea and imidazolinone herbicides. The mechanism conferring resistance to glyphosate identified in this population was gene amplification, with increased EPSPS copy number - between 50 and 179 more copies in BR-R. Besides, two target-site mutations were identified in the ALS gene sequencing, W574L and S653N, conferring resistance to sulfonilureas and imidazolinones. The weed control experiment, overal, herbicide tank mixtures achieved higher levels of control. Therefore, this research confirms the introduction of A. palmeri to Brazil, as well as its multiple resistance to EPSPS and ALS inhibitor herbicides. Its control is more efficient with herbicide mixtures, which guarantees more susteinable use of the new herbicide-tolerant crop technologies.

Abstract not available

Determining the properties of mutations is fundamental to understanding the mechanisms of adaptive evolution. The major goal of this thesis is to investigate the mechanisms of bacterial adaptation to new environments using experimental evolution. Different types of mutations were under investigations with a particular focus on genome rearrangements. Adaptive evolution experiments were focused on the development of bacterial resistance to antibiotics. In paper I, we performed stochastic simulations to examine the role of gene amplification in promoting the establishment of new gene functions. The results show that gene amplification can contribute to creation of new gene functions in nature. In paper II, the evolution of β-lactam resistance was studied by evolving S. typhimurium carrying a β-lactamase gene towards increased resistance against cephalosporins. Our results suggest that gene amplification is likely to provide an immediate solution at the early stage of adaptive evolution and subsequently facilitate further stable adaptation. In paper III, we isolated spontaneous deletion mutants with increased competitive fitness, which indicated that genome reduction could be driven by selection. To test this hypothesis, independent lineages of wild type S. typhimurium were serially passaged for 1000 generations and we observed fixation of deletions that significantly increased bacterial fitness when reconstructed in wild type genetic background. In paper IV, we developed a new strategy combining 454 pyrosequencing technology and a ‘split mapping’ computational method to identify unique junction sequences formed by spontaneous genome rearrangements. A high steady-state frequency of rearrangements in unselected bacterial populations was suggested from our results. In paper V, the rates, mechanisms and fitness effects of colistin resistance in S. typhimurium were determined. The high mutation rate and low fitness costs suggest that colistin resistance could develop in clinical settings. In paper VI, a novel Metallo-β-lactamase (MBL) with low resistance against β-lactam antibiotics was employed as the ancestral protein in a directed evolution experiment to examine how an enzyme evolves towards increased resistance. For most isolated mutants, in spite of their significantly increased resistance, both mRNA and protein levels were decreased as compared with the parental protein, suggesting that the catalytic activity had increased.