Orientador: Marcelo Menossi Teixeira / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-04T02:01:30Z (GMT). No. of bitstreams: 1 CostaNetto_AntonioPaulinoda_D.pdf: 960397 bytes, checksum: 250e582a4c1b0747546c9f611a5248d2 (MD5) Previous issue date: 2004 / Resumo: O óxido nítrico (NO) é uma espécie reativa de nitrogênio relativamente instável e possui grande versatilidade. Ele atua como mensageiro secundário, sendo inclusive citotóxico dependendo da sua concentração. Ações do NO como mensageiro secundário são descritas na neurotransmissão, regulação da circulação sanguínea e de processos inflamatórios, principalmente em mamíferos onde a enzima NO-sintase (NOS) foi descoberta. Em plantas, o NO possui importante papel na interação planta-patógeno, na expansão foliar e redução da velocidade de amadurecimento ou senescência de frutos e flores e no processo de apoptose, entre outros. Recentemente duas isoformas de NOS de plantas foram caracterizadas em Arabidopsis thaliana e pouco se sabe da modulação da expressão gênica por NO em plantas. Este trabalho relata a clonagem e caracterização de um gene que codifica uma NOS de A. thaliana e a modulação da expressão gênica em cana-deaçúcar em resposta GSNO, um doador de NO. partir de seqüências depositadas no banco de dados do NCBI, foram sintetizados oligonucleotídeos específicos. A partir de reações de transcrição reversa e PCR o gene foi clonado e seqüenciado, sendo denominado HHP. O gene é mais expresso em folhas e flores e é induzido quando plantas de A. thaliana são submetidas a baixa temperatura. A localização subcelular da proteína foi determinada via fusão com GFP, sendo predominantemente plastídica. Plantas de A. thaliana superexpressando o gene HHP apresentaram uma melhora acentuada na resposta ao frio. O efeito do NO na expressão gênica em cana-de-açúcar foi avaliado empregando macroarranjos de DNA. Foi detectado um aumento na expressão de alguns genes como por exemplo minotransferases e peroxidases, além de proteínas com função ainda desconhecidas. Foi também demonstrada a influência dos plásticos que selam membranas com macroarranjos de DNA, onde compostos a partir de polietileno podem bloquear a emissão de radiação e diminuir o número de spots detectados, o que significa um menor número de genes avaliados / Abstract: Nitric oxide (NO) is a reactive nitrogen species relatively unstable that has great versatility. NO acts as a secondary messenger and may be even cytotoxic, depending on its concentration. Actions of the NO as secondary messenger have been described in neurotransmission, regulation of the blood circulation and inflammatory processes mainly in mammals where the enzyme NO-synthase (NOS) was discovered. In plants, NO has important role in plant-pathogen interactions, in foliar expansion and in the maturation and sencescence of fruits and flowers. Recently two NO isoforms have been characterized in Arabidopsis thaliana, but we are far from a complete understanding of the NO effects on gene expression. This work reports the cloning and characterization of a NOS isoform from A. thaliana and the modulation of gene expression in sugarcane by NO donors. Gene-specific oligonucleotides were designed based on the sequence of an animal NO-synthase deposited in the NCBI. After reverse-transcription and PCR the open reading frame was cloned and the gene was named HHP. The highest levels of gene expression were observed in leaves and flowers. HHP was induced by cold stress. The subcelular localization of the protein, determined using a GFP fusion, was predominantly plastidic. Transgenic A. thaliana superexpressing the HHP gene were more tolerant to cold stress. The effect of NO on sugarcane cells was evaluated using DNA macroarrays. Several genes were found to be modulated by NO, such as those encoding an aminotransferase and a peroxidase. Surprisingly, most of them encode proteins with unknown function. The influence of plastics used to seal macroarray membranes was also evaluated. It was found that some of them block the radiation transmittance, reducing the number of detected spots and consequently the number of genes evaluated in the assay. These findings indicate that the choice of the sealing plastic is very important to obtain the maximum information from nylon arrays / Doutorado / Bioquimica / Doutor em Biologia Funcional e Molecular

Memoria para optar al Título Profesional de Médico Veterinario / La resistencia antimicrobiana es un fenómeno de gran importancia a nivel mundial, debido a sus consecuencias en la salud tanto humana como animal y también por las consecuencias económicas que genera. Entre las bacterias que presentan multirresistencia se encuentra Salmonella spp., patógeno distribuido mundialmente, generalmente transmitido por los alimentos, pero también por contacto directo o indirecto con su reservorios animales. Dentro de estos, adquieren relevancia los reptiles por ser portadores asintomáticos de la bacteria, constituyendo un reservorio que cada vez adquiere mayor importancia debido a la creciente popularidad que presentan como mascota en las familias actuales. En Chile existen escasos estudios sobre resistencia antimicrobiana en cepas de Salmonella spp. aisladas de reptiles que asocien genes a la resistencia fenotípica, razón por la cual, el objetivo del presente estudio apunta a la detección de genes de resistencia antimicrobiana en cepas de Salmonella spp. aisladas de reptiles en cautiverio, en la Región Metropolitana en Chile. Para ello, se realizó una prueba de susceptibilidad antimicrobiana por el método de Kirby-Bauer y posteriormente, mediante la técnica de PCR, se buscaron 11 genes de resistencia en las cepas en análisis, independiente de su fenotipo. Dentro de los genes a detectar, 3 de ellos están asociados a resistencia a beta-lactámicos (blaTEM, blaOXA y blaPSE-1), 4 a resistencia frente a estreptomicina (aadA1, aadA2, strA y strB), 3 para resistencia a tetraciclinas (tet(A), tet(B), tet(C)) y un gen que confiere resistencia a cloranfenicol (cmlA). Se observó una elevada sensibilidad fenotípica, donde el 23,5% de las cepas fue resistente a estreptomicina. De las 34 cepas analizadas, sólo se detectó el gen blaTEM en un 61,7%. El resto de las cepas resultaron negativas a la detección de los otros genes en análisis. La alta sensibilidad detectada puede deberse a fenómenos de curación plasmidial, mutaciones cromosomales, regulación de la expresión génica, entre otros, como mecanismo de restauración del fitness bacteriano en cepas conservadas en un medio carente de presión selectiva. Se concluye que las cepas analizadas presentan elevada sensibilidad fenotípica frente a beta-lactámicos, tetraciclinas, cloranfenicol y estreptomicina, encontrando sólo cepas portadoras del gen blaTEM. / Antimicrobial resistance is a phenomenon of great importance worldwide because of its impact on both human and animal health and the economic consequences it generates. Among resistant bacteria is Salmonella spp., worldwide distributed pathogen, usually transmitted by food, but also by direct or indirect contact with animal reservoirs. Within these, reptile become relevant because they are asymptomatic carriers of the bacteria, forming a reservoir that increasingly becomes more important due to the growing popularity as pets in today's families. In Chile there are few studies on antimicrobial resistance in strains of Salmonella spp. isolated from reptiles that associated genes to phenotypic resistance, reason why the aim of this study points to the detection of antimicrobial resistance genes in strains of Salmonella spp. isolated from reptiles in captivity, in Región Metropolitana in Chile. For this, an antimicrobial susceptibility test, by Kirby-Bauer method was performed and subsequently, by PCR, 11 resistance genes were searched in strains tested, regardless of their phenotype. Among genes to detect, 3 of them are associated with resistance to beta-lactam (blaTEM, blaOXA and blaPSE-1), 4 to resistance to streptomycin (aadA1, aadA2, strA and strB), 3 for tetracycline resistance (tet(A), tet(B), tet(C)) and a gene conferring chloramphenicol resistance (cmlA). High phenotypic susceptibility was observed, where 23,5% of the strains were streptomycin resistant. Of the 34 strains tested, only blaTEM gene was detected in 61,7%. The remaining strains were negative for detection of the other genes analyzed. High sensitivity detected may be due to a phenomenon of plasmidial healing, chromosomal mutations, gene expression regulation, among others, as a mechanism for restoring the bacterial fitness of strains preserved in a medium lacking of selective pressure. It is concluded that strains tested possess high phenotypic sensitivity to beta-lactams, tetracyclines, chloramphenicol and streptomycin, finding strains carrying only the blaTEM gene.

Orientador: Laura M. M. Ottoboni / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-07-19T20:49:55Z (GMT). No. of bitstreams: 1 SouzaFilho_GoncaloApolinariode_M.pdf: 4023775 bytes, checksum: e56b411d7a4dbe56f49298bfbd3061f5 (MD5) Previous issue date: 1994 / Mestrado / Genetica de Plantas / Mestre em Ciências Biológicas

Orientador: Angelo Berchieri Junior / Resumo: Salmonella Heidelberg (SH) é um dos sorovares do gênero Salmonella capaz de infectar tanto seres humanos quanto animais. Surtos infecciosos em seres humanos estão relacionados principalmente à ingestão de produtos de origem avícola e o tratamento é realizado com o uso de antimicrobianos. Entretanto, estas drogas também são utilizadas na produção avícola como melhoradores de desempenho, o que resulta em resistência bacteriana em SH, já relatada em literatura. Com isso, o objetivo deste estudo foi caracterizar 62 isolados de SH quanto ao fenótipo de resistência a 20 antimicrobianos; identificar o genótipo de resistência aos β-lactâmicos por meio da PCR; e avaliar a similaridade dos isolados utilizando as metodologias ERIC-PCR, REP-PCR, BOX-PCR e PFGE. Todas as estirpes de SH foram confirmadas por PCR e submetidas aos testes de sensibilidade a 20 antimicrobianos. A resistência foi observada em 16 dos 20 antimicrobianos utilizados, ressaltando a elevada resistência aos β-lactâmicos (CEF, FOX, AMP, CTX, AMX, AMC e IMP) com a identificação de 41 estirpes multirresistentes. A partir da PCR identificou-se genes codificadores de enzimas ESBL e AmpC, havendo predominância do gene blaCMY-2. O PFGE demonstrou ser a metodologia com a maior diversidade em comparação as demais utilizadas, com a maioria das estirpes sendo agrupadas de acordo com a fonte de isolamento. A partir dos resultados deste estudo é possível observar a diversidade dos isolados de SH no Brasil albergando genes AmpC e a... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Salmonella Heidelberg (SH) is one of the serovars of the Salmonella genus capable of infecting both humans and animals. Infectious outbreaks in humans are mainly related to the consumption of products of poultry origin and the treatment is carried out with the use of antimicrobials. However, these drugs are also used in poultry production as performance enhancers, which results in bacterial resistance in SH, already reported in the literature. Thus, the aim of this study was to characterize 62 SH isolates regarding the phenotype of resistance to 20 antimicrobials; identify the genotype of resistance to β-lactams by means of PCR; and to evaluate the similarity of the isolates using the ERIC-PCR, REP-PCR, BOX-PCR and PFGE methodologies. All strains of SH were confirmed by PCR and subjected to sensitivity tests to 20 antimicrobials. Resistance was observed in 16 of the 20 antimicrobials used, highlighting the high resistance to β-lactams (CEF, FOX, AMP, CTX, AMX, AMC and IMP) with the identification of 41 multidrug resistant strains. From the PCR, genes encoding ESBL and AmpC enzymes were identified with a predominance of the blaCMY-2 gene. The PFGE proved to be the methodology with the greatest diversity compared to the others used with the majority of strains being grouped according to the isolation source. From the results of this study, it is possible to observe the diversity of SH isolates in Brazil harboring AmpC genes and presenting different profiles of phenotypic multid... (Complete abstract click electronic access below) / Mestre

Mycobacterium tuberculosis (Mtb) is the leading cause of death from an infectious disease. The success of the pathogen lies in its ability to subvert hostile intracellular macrophage environment. We performed genome-wide transcriptional deep sequencing on total RNA in murine bone marrow-derived macrophages (BMDM) infected with hypervirulent Beijing strain (HN878) in an extensive time kinetic manner using single molecule sequencer and cap analysis gene expression (CAGE) technique. CAGE analysis revealed nearly 36000 unique RNA transcripts with approximately 16000 are not unannotated to a specific gene. This thesis addressed global changes in RNA expression levels in macrophages infected with Mtb in a time kinetic manner to pinpoint novel host protection and immune evasion genes and elucidate the role of these genes in vitro macrophage assays and in vivo knockout mouse studies. The data in this thesis showed that basic leucine zipper transcription factor 2 (Batf2) was an important factor that regulates inflammatory responses in Mtb infection. Deletion of Batf2 led to the survival of mice with reduced lung inflammation and histopathology due to reduced recruitment of inflammatory macrophages. We also showed that Batf2 was highly expressed in peripheral blood from adolescents who progressed from infection to tuberculosis disease and a predictive human biomarker for tuberculosis disease. In contrast to Batf2, we showed that Protein Kinase C-delta (PKC-δ) deficient mice are highly susceptible to tuberculosis and human lung proteomics dataset revealed that PKC-δ was highly upregulated in the necrotic and cavitory regions of human granulomas in multi-drug resistant subjects. PKC-δ deficient mice had a significant reduction in alveolar macrophages and dendritic cells, reduced accumulation of lipid bodies and serum fatty acids. In vitro experiments showed that PKCδ was required for optimal killing effector functions which were independent of phagosome maturation and autophagy in primary murine macrophages. Our studies suggested that these novel genes play a role in the immune response to Mtb and should be studied more thoroughly to evaluate their potential in possible TB interventions.

Indiana University-Purdue University Indianapolis (IUPUI) / Chlamydia trachomatis is the leading cause of bacterial sexually transmitted disease. Chlamydia spp. are all obligate intracellular organisms that undergo a biphasic developmental cycle within a vacuole termed the inclusion. Infectious, non metabolically active elementary bodies (EBs) are endocytosed and differentiate into non infectious, metabolically active reticulate bodies (RBs) before re-differentiating back into EBs. The chlamydial factors that mediate these differentiation events are mostly unknown. Comparative genomics revealed that Chlamydia spp. have small, highly conserved genomes, suggesting that many of their genes may be essential. Genetic manipulation strategies for Chlamydia spp. are in their infancy, and most of these cannot be used to inactivate essential genes. We generated a clonal ethyl methanesulfonate (EMS)-mutagenized C. trachomatis library and screened it for temperature sensitive (TS) mutants that produced fewer inclusions at either 32°C or 40°C compared to 37°C. Because EMS mutagenesis elicited multiple mutations in most of the library isolates, we also developed a novel lateral gene transfer strategy for mapping mutations linked to TS phenotypes. We identified TS alleles of genes that are essential in other bacteria and that are involved in diverse biological processes including DNA replication, protein synthesis, carbohydrate metabolism, fatty acid biosynthesis, and energy generation, as well as in highly conserved chlamydial hypothetical genes. TS DNA polymerase (dnaEts) and glutamyl-tRNA synthestase (gltXts) mutants were characterized further. Both the dnaEts and gltXts mutants failed to replicate their genomes at 40°C but exhibited unique signs of stress. Chlamydial DNA replication begins by 12 hpi and protein synthesis begins by 2 hpi. However, inclusion expansion and replication of both of the mutants could be rescued by shifting to them to 37°C prior to mid-late development. Since gltXts is likely unable to produce aminoacyl-tRNAs at 40°C, our observation suggests that de novo chlamydial translation uses a pre-existing pool of aminoacyl-tRNA in EBs. Genetic suppressor analysis indicated that the inability of the dnaEts mutant to replicate its genome at 40°C might be linked to an inability of mutant DnaE to bind the DNA template. The tools and mutants we have identified will be invaluable assets for investigating many essential aspects of chlamydial biology.

Basic helix-loop-helix (bHLH) proteins belong to a class of transcription factors that are critical regulators of development, cell growth and differentiation. One particular family member includes the products of the E2A gene, E12 (Pan-2) and E47 (Pan-1), ubiquitous transcription factors localized in the nucleus. E12 and E47 gene products are generated by alternative RNA splicing. E12 and E47 proteins have been implicated as transcriptional regulators of the rat I insulin gene, immunoglobulin light and heavy chain genes and several muscle-specific genes. To delineate the role ofE2A proteins in directing insulin gene transcription, we have characterized an anti-E2A polyclonal antiserum which recognizes both E12 and E47 and used this reagent to study E2A proteins in the pancreas. In these studies, we have demonstrated that the anti-E2A polyclonal antiserum is highly specific for E2A proteins in a variety of different cell lines representing different tissues. Furthermore, using this immunohistochemical tool, we have demonstrated that E2A proteins are posttranslationally modified in beta cells, insulin producing cells in the pancreas. We also provide evidence that a posttranslationally modified form of E2A protein is involved in glucose-induced insulin gene transcription in beta cells. Lastly, we provide evidence that E2A proteins are associated with other bHLH proteins or other non-bHLH proteins in pancreatic beta cells.

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