Made available in DSpace on 2014-06-11T19:34:57Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-07-02Bitstream added on 2014-06-13T20:25:34Z : No. of bitstreams: 1 gonzalez_to_dr_jabo.pdf: 459034 bytes, checksum: 09affcd8cd969944ecfadd924ad3cffc (MD5) / Universidade Central da Venezuela / A diversidade genética de quinze estirpes de rizóbios isoladas do feijoeiro, provenientes de várias localidades do Brasil e da Venezuela, foi determinada pelo seqüenciamento do gene 16S rRNA. Selecionaram-se as duas melhores estirpes de cada país, com base em nodulação, produção de massa seca e de nitrogênio total em plantas de feijão, e compararam-se as suas produtividades e nodulação no feijoeiro, com duas estirpes comerciais, CIAT-899 e PRF-81, em um solo Latossolo Vermelho-Escuro da região de Jaboticabal SP. Determinou-se ainda a diversidade genética das populações nativas do solo e das quatro estirpes, pelo seqüenciamento do espaço intergênico, entre os genes 16S e 23S rRNA. Experimentos foram conduzidos em blocos casualizados com três repetições, em casa de vegetação, na UNESP, em 2007. O primeiro foi realizado em tubetes com vermiculita e quinze tratamentos: sete diluições seriadas do solo de 10-1 a 10-7, as quatro estirpes, as comerciais e duas testemunhas com e sem nitrogênio. Das colônias isoladas extraiu-se o DNA genômico e realizou-se o seqüenciamento do espaço intergênico. O segundo experimento foi realizado em vasos contendo solo e treze tratamentos: as quatro estirpes isoladamente e misturadas com a estirpe PRF- 81, as comerciais, a mistura delas, e duas testemunhas com e sem nitrogênio. Houve coincidência entre os marcadores moleculares do gene 16S rRNA e o espaço intergênico na identificação das espécies. Encontraram-se estirpes tão produtivas quanto as comercias. A mistura com a estirpe PRF-81 não incrementou a produtividade e produziu um efeito antagônico com a estirpe LBMP-12BR. A população nativa do solo foi identificada como Rhizobium sp., sendo ineficiente na fixação de nitrogênio. Há estirpes promissoras que mostram uma resposta diferencial quando são misturadas nos inoculantes. / The genetic diversity of 15 rhizobia strains isolated from common beans grown in Brazil and Venezuela was determined by sequencing the 16S rRNA gene. Two strains were selected from each country for further study based on nodulation, dry weight, and total nitrogen in the common bean plants, and productivity and nodulation on the common bean of these strains were evaluated in Latossolo Vermelho Escuro soil of Jaboticabal SP, in comparison to two commercial strains, CIAT-899 and PRF-81. The genetic diversity of the native soil population and the four strains was determined by sequencing of the intergenic space between the 16S and 23S rRNA genes. In 2007, experiments were carried out under glass house conditions at the UNESP. The first was conducted in tubs with vermiculite, and 15 treatments were examined: seven soil serial dilutions (10-1 to 10-7), the four novel strains, two commercial strains, and two controls with and without nitrogen. From the pure isolated colonies, DNA was extracted, and the intergenic space was sequenced. The second experiment was conducted in pots filled with soil, and 13 treatments were examined: the four strains alone and in mixture with the PRF-81 strain, the two commercial strains alone and in mixture, and two controls with and without nitrogen. There was coincidence between the 16S rRNA gene and the intergenic space molecular markers for species identification. Strains that had productivity values equivalent to the commercial strains were identified. The mixture of PRF-81 strain to each one of the four strains did not increase productivity and produced an antagonistic effect on the LBMP-12BR strain. The native soil population was identified as Rhizobium sp. and was found to be inefficient in nitrogen fixation. This study identified promising new Rhizobium strains that exhibit differential responses in mixtures on inoculants.

Le ribosome est une machine biomoléculaire primordiale pour la survie de tout organisme du fait de son rôle central au sein de la synthèse protéique. La caractérisation des interactions avec ses nombreux partenaires est un élément crucial pour mieux comprendre les mécanismes de la traduction et de son inhibition chez les eucaryotes et procaryotes. Cette inhibition est d’ailleurs une stratégie utilisée par beaucoup d’antibiotiques ciblant le ribosome pour lutter contre les infections bactériennes. La compréhension de leur mode d’action est devenue une priorité mondiale pour faire face au problème de la résistance bactérienne. Chez les eucaryotes, une autre stratégie est employée par les virus pour bloquer et s’approprier la machinerie traductionnelle de l’hôte grâce à des structures d’ARN non codant (IRES) capables de recruter directement le ribosome. Bien que largement caractérisés, peu de données thermodynamiques et cinétiques sont disponibles concernant ces deux systèmes d’interaction avec le ribosome. Mon projet a pour vocation d’utiliser des approches biophysiques innovantes afin de compléter les études sur les interactions du ribosome d’E. coli avec les macrolides, et du ribosome de S. cerevisiae avec l’IRES intergénique du CrPV. / The ribosome is a biomolecular machine essential for the survival of any organism due to its central role in protein synthesis. The characterization of its interactions with its many partners is a crucial element in better understanding the mechanisms of translation and inhibition in eukaryotes and prokaryotes. Inhibition of translation is a strategy used by many ribosome-targeting antibiotics to fight bacterial infections. Understanding their mode of action has become a global priority in addressing the problem of bacterial resistance. In eukaryotes, another strategy is used by viruses to block and appropriate the host's translational machinery through non-coding RNA structures (IRES) capable of directly recruiting the ribosome. Although widely characterized, few thermodynamic and kinetic data are available for these two ribosome interaction systems. My project is intended to use innovative biophysical approaches in order to provide an original view of the interactions of the E. coli ribosome with macrolides, and of the S. cerevisiae ribosome with the intergenic IRES of the CrPV.

Flera olika arter av mjölksyrabakterier som ingår i släktena Lactobacillus och Bifidobacterium har hittats hos bin och i deras honung. Idag finns ingen effektiv metod för identifiering av bakterierna. Syftet med detta projekt är att utveckla en metod för snabb identifiering genom att hitta lämpliga primers till olika mjölksyrabakterier och därmed få fram en Polymeraskedjereaktion (PCR) metod. Ribosomal ribonukleinsyra (rRNA) generna eller 16S-23S rRNA intergenic spacer region (ISR) används ofta vid design av primers, som därefter används i PCR för att identifiera olika bakterier. Deoxiribonukleinsyra (DNA) visualiseras i agarosgelen med hjälp av SYBRgreen I som fluorescens på ultraviolett (UV)-ljusbord. I detta projekt har 16S rRNA och 16S-23S rRNA ISR amplifierats i enkel PCR och multiplex PCR och visualiserats i agarosgel i försök att identifiera mjölksyrabakterierna. 16S rRNA har visat sig ha mycket liten variation mellan bakterierna och ansågs därför inte lämplig att använda för identifiering av närbesläktade arter. 16S-23S rRNA ISR visade större variation, fram för allt mellan lactobacillerna och bifidobakterierna. Gruppering av bakterierna med hjälp av multiplex PCR gjordes med viss framgång, med undantag av några bakterier som inte hamnade i den förväntade gruppen. Dock behövs fler försök för att stödja dessa resultat. / Several different lactic acid bacterium (LAB) species from the genera Lactobacillus and Bifidobacterium was discovered in bees and in their honey. Today there is no rapid and reliable method to identify these LAB. Therefore a rapid polymerase chain reaction (PCR) method to identify the LAB is needed. The aim of this project is to find primers suitable for the different LAB. Ribosomal ribonucleic acid (rRNA) genes or 16S-23S rRNA intergenic spacer region (ISR) are often used to designing of primers followed by PCR assays, for identification of different bacteria. To visualize deoxyribonucleic acid (DNA) in agarose gels, SYBRgreen I was used as fluorescence and then viewed under ultraviolet (UV) light. In this project the 16S rRNA and 16S-23S rRNA ISR was used as a target in a PCR and a multiplex PCR amplification. The PCR product was analyzed in agarose gel in an attempt to identify the LAB. 16S rRNA sequence have to little variation and is not suitable to identify closely related species. 16S-23S rRNA ISR sequence exhibits greater variations, especially between Lactobacillus and Bifidobacterium. Differentiation of the bacteria into groups by multiplex PCR was done with good result, except for some of the bacteria that did not end up in the expected group. More studys is needed to support these results.

No description available.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Land-use change and soil type can have significant impact on microbial communities of soil. The Pampa biome in recent decades has undergone severe changes in landscape due to landuse change, mainly for the introduction of exotic tree plantation and croplands. Different landuse in Pampa biome were evaluated to determine the effect on the structure of soil microbial communities. Furthermore, due to the presence of various soil types present in this biome, we investigated whether different soil type harbor different microbial communities. Soil samples were collected at two sites with different land-uses (native grassland, native forest, exotic tree plantation and cropland) and in a typical toposequence in Pampa biome formed by Paleudult, Albaqualf and alluvial soils. The structure of soil microbial community (archaeal and fungal) was evaluated by RISA and soil functional capabilities were measured by microbial biomass carbon and metabolic quotient. We detected different patterns in fungal and archaeal community driven by land-use change and soil type showing that both factors are significant drivers of microbial community structure and activity. Acacia and Eucalyptus afforestation presented the most dissimilar communities when compared with natural vegetation. Although differences in the communities were detected, the soils tested shared most of the taxonomic unities and only a proportion of the community suffers changes caused by human interference. / A mudança do uso da terra e o tipo de solo podem exercer impactos significantes sobre a comunidade microbiana do solo. O bioma Pampa Brasileiro, nas últimas décadas, tem sofrido severas mudanças na paisagem devido à mudança no uso da terra, principalmente pela introdução de plantações de árvores exóticas e pelos cultivos agrícolas. Diferentes usos do solo no bioma Pampa foram avaliados para determinar o efeito sobre a estrutura das comunidades microbianas do solo. Além disso, devido à presença de vários tipos de solo presentes neste bioma, foi investigado se diferentes tipos de solos abrigam diferentes comunidades microbianas. Amostras de solo foram coletadas em duas áreas com diferentes usos do solo (pastagem nativa, mata nativa, plantações de árvores exóticas e cultivo agrícola) e em uma topossequência típica no bioma Pampa formado por Argissolo, Planossolo e solos aluviais. A estrutura das comunidades microbianas do solo (arqueas e fungos) foi avaliada por RISA e capacidades funcionais do solo foram mensuradas através de carbono da biomassa microbiana e quociente metabólico. Diferentes padrões foram detectados nas comunidades de fungos e arqueas influenciados pela mudança no uso da terra e pelo tipo de solo, mostrando que ambos são importantes fatores da estrutura e atividade da comunidade microbiana. Florestamentos de acácia e eucalipto apresentaram as comunidades mais diferentes quando comparados com a vegetação natural. Embora diferenças nas comunidades foram detectadas, os diferentes usos e tipos de solos avaliados compartilham grande parte das unidades taxonômicas e mostram que apenas uma parte da comunidade sofre alterações causadas pela interferência humana.

The TRIB1 locus (8q24.13) is a novel locus associated with plasma TGs and CAD risk. Trib1 is a regulator of MAPK activity, and has been shown to regulate hepatic lipogenesis and VLDL production in mice. However, the functional relationship between common SNPs at the TRIB1 locus and plasma lipid traits is unknown; TRIB1 has not been identified as an eQTL. This cluster of SNPs falls within an intergenic region 25kb to 50kb downstream of the TRIB1 coding region. By phylogenetic footprinting analysis and DNA genotyping, we identified an evolutionarily conserved region (CNS1) within the risk locus that harbours two common SNPs in tight LD with GWAS risk SNPs and significantly associated with CAD. We investigated the regulatory function of CNS1 by luciferase reporter assays in HepG2 cells and demonstrate that this region has promoter activity. In addition, the rs2001844 risk allele significantly reduces luciferase activity, suggesting that altered expression of the EST-based gene may be associated with plasma TGs. We identified an EST within the risk locus directly downstream of CNS1. We performed 5'/3' RACE using HepG2 RNA, identified multiple variants of this EST-based gene, and confirmed its transcription start site within CNS1. We hypothesize that this EST is a long noncoding RNA due to low abundance, poor conservation, and absence of significant ORF. Over-expression of a short variant implicates its function in the regulation of target gene transcription, although the mechanism of action remains unknown. We conclude that the risk locus at 8q24.13 harbours a novel EST-based gene that may explain the relationship between GWAS SNPs at this locus and plasma lipid traits.

The TRIB1 locus (8q24.13) is a novel locus associated with plasma TGs and CAD risk. Trib1 is a regulator of MAPK activity, and has been shown to regulate hepatic lipogenesis and VLDL production in mice. However, the functional relationship between common SNPs at the TRIB1 locus and plasma lipid traits is unknown; TRIB1 has not been identified as an eQTL. This cluster of SNPs falls within an intergenic region 25kb to 50kb downstream of the TRIB1 coding region. By phylogenetic footprinting analysis and DNA genotyping, we identified an evolutionarily conserved region (CNS1) within the risk locus that harbours two common SNPs in tight LD with GWAS risk SNPs and significantly associated with CAD. We investigated the regulatory function of CNS1 by luciferase reporter assays in HepG2 cells and demonstrate that this region has promoter activity. In addition, the rs2001844 risk allele significantly reduces luciferase activity, suggesting that altered expression of the EST-based gene may be associated with plasma TGs. We identified an EST within the risk locus directly downstream of CNS1. We performed 5'/3' RACE using HepG2 RNA, identified multiple variants of this EST-based gene, and confirmed its transcription start site within CNS1. We hypothesize that this EST is a long noncoding RNA due to low abundance, poor conservation, and absence of significant ORF. Over-expression of a short variant implicates its function in the regulation of target gene transcription, although the mechanism of action remains unknown. We conclude that the risk locus at 8q24.13 harbours a novel EST-based gene that may explain the relationship between GWAS SNPs at this locus and plasma lipid traits.

The TRIB1 locus (8q24.13) is a novel locus associated with plasma TGs and CAD risk. Trib1 is a regulator of MAPK activity, and has been shown to regulate hepatic lipogenesis and VLDL production in mice. However, the functional relationship between common SNPs at the TRIB1 locus and plasma lipid traits is unknown; TRIB1 has not been identified as an eQTL. This cluster of SNPs falls within an intergenic region 25kb to 50kb downstream of the TRIB1 coding region. By phylogenetic footprinting analysis and DNA genotyping, we identified an evolutionarily conserved region (CNS1) within the risk locus that harbours two common SNPs in tight LD with GWAS risk SNPs and significantly associated with CAD. We investigated the regulatory function of CNS1 by luciferase reporter assays in HepG2 cells and demonstrate that this region has promoter activity. In addition, the rs2001844 risk allele significantly reduces luciferase activity, suggesting that altered expression of the EST-based gene may be associated with plasma TGs. We identified an EST within the risk locus directly downstream of CNS1. We performed 5'/3' RACE using HepG2 RNA, identified multiple variants of this EST-based gene, and confirmed its transcription start site within CNS1. We hypothesize that this EST is a long noncoding RNA due to low abundance, poor conservation, and absence of significant ORF. Over-expression of a short variant implicates its function in the regulation of target gene transcription, although the mechanism of action remains unknown. We conclude that the risk locus at 8q24.13 harbours a novel EST-based gene that may explain the relationship between GWAS SNPs at this locus and plasma lipid traits.

The TRIB1 locus (8q24.13) is a novel locus associated with plasma TGs and CAD risk. Trib1 is a regulator of MAPK activity, and has been shown to regulate hepatic lipogenesis and VLDL production in mice. However, the functional relationship between common SNPs at the TRIB1 locus and plasma lipid traits is unknown; TRIB1 has not been identified as an eQTL. This cluster of SNPs falls within an intergenic region 25kb to 50kb downstream of the TRIB1 coding region. By phylogenetic footprinting analysis and DNA genotyping, we identified an evolutionarily conserved region (CNS1) within the risk locus that harbours two common SNPs in tight LD with GWAS risk SNPs and significantly associated with CAD. We investigated the regulatory function of CNS1 by luciferase reporter assays in HepG2 cells and demonstrate that this region has promoter activity. In addition, the rs2001844 risk allele significantly reduces luciferase activity, suggesting that altered expression of the EST-based gene may be associated with plasma TGs. We identified an EST within the risk locus directly downstream of CNS1. We performed 5'/3' RACE using HepG2 RNA, identified multiple variants of this EST-based gene, and confirmed its transcription start site within CNS1. We hypothesize that this EST is a long noncoding RNA due to low abundance, poor conservation, and absence of significant ORF. Over-expression of a short variant implicates its function in the regulation of target gene transcription, although the mechanism of action remains unknown. We conclude that the risk locus at 8q24.13 harbours a novel EST-based gene that may explain the relationship between GWAS SNPs at this locus and plasma lipid traits.

A protein, B0238.11, was identified in a yeast one-hybrid screen to bind to the ribosomal intergenic spacer region (IGS) of Caenorhabditis elegans. Proteins interacting with this region of the DNA have been implicated in ribosome biogenesis in other model organisms, so it is also possible that B0238.11 plays a role in RNA transcription by interacting with RNA polymerase I or other transcription machinery. Thus, the goal of this study was to further characterize the structure and function of B0238.11. I used yeast two-hybrid experiments to identify proteins that interact with B0238.11 within the nucleus. RPS-0, K04G2.2, DPY-4, EFT-3, PAL-1, and B0238.11, itself, were found to bind to B0238.11. Additionally, I analysed the amino acid sequence of B0238.11 using in silico bioinformatics methods to determine its structure and putative function and also to identify and characterize the other interacting proteins. I found that B0238.11 contains a high-mobility group box domain, which is also found in HMO1P in yeast and UBF in vertebrates. These other proteins also bind to the IGS, are known to form homodimers and have been implicated in the initiation of ribosomal RNA transcription. Here I scrutinize the validity of the interaction between each protein and B0238.11. I conclude that B0238.11 is likely to be a C. elegans homolog of UBF and present an updated interactome map for B0238.11. / Synopsis: I carried out yeast two-hybrid assay to find proteins interacting with B0238.11 (O16487_CAEEL). I found that this protein's DNA-binding profile and protein interaction profile mimic other HMG-box containing proteins UBF and HMO1P which are involved in ribosomal RNA transcription initiation. Acknowledgements: I would like to thank my supervisor, Dr. Teresa J. Crease, for not only giving me the opportunity to investigate an interesting topic in Molecular Biology, but also for her patient guidance, encouragement and sound advice. I feel extremely lucky to have a supervisor who cared so much about my work, who responded to my questions and queries so promptly, and was always available to discuss project and career related matters. I would also like to thank Dr. Todd Gillis and Dr. Terry Van Raay for their careful consideration of this project and timely constructive criticisms that helped shape my project. I would like to thank all the members of my committee for helping me see things from different perspectives and helping me develop and critical and mature understanding of the scientific process. I must also express my gratitude to Dr. Robin Floyd for allowing me to build upon his work and Dr. Marian Walhout, at the University of Massachusetts, for providing the Caenorhabditis elegans complimentary DNA library. A large part of this project would not have been possible without the people at the genomics facility in the Department of Integrative Biology, I commend their professionalism and punctuality in delivering results. Completing this work would have been all the more difficult were it not for the support and friendship provided by my peers Shannon Eagle, Tyler Elliott, Nick Jeffery, Joao Lima, Sabina Stanescu, Fatima Mitterboeck and Paola Pierossi. And finally, I would like to thank my parents and siblings Sara Omar and Ali Omar for their continued support through good times and bad, and letting me use their laptops when mine broke down.