O filo Actinobacteria é, atualmente, considerado um dos maiores filos pertencentes ao domínio Bacteria. Este filo pode representar até 30% da comunidade microbiana do solo. Os organismos do filo Actinobacteria tem sido caracterizados como produtores de antibiótico e degradadores de substâncias complexas como os alcanos, presente em diversas substâncias encontradas em plantas. Em relação à degradação dos alcanos já tem sido comprovado que o gene alkB (alcano hidroxilase), presente no filo Actinobacteria, está relacionado a sua degradação. Porém, as comunidades de tais organismos podem ser alteradas por mudanças no uso do solo, que no Brasil ocorrem extensivamente devido a implantação de monoculturas. A cultura de cana-de-açúcar, que vem se expandido anualmente, se mostra como uma das culturas que podem alterar a microbiota do solo. Os diferentes sistemas de colheita de cana-de-açúcar, com e sem a queima da palhada, podem alterar a microbiota do solo. Assim, considerando o papel do filo Actinobacteria na ciclagem de matéria orgânica, além da sua importância biotecnológica, este trabalho avaliou como os diferentes sistemas de colheita da cana-de-açúcar influenciam as comunidades rizosféricas de Actinobacteria e de bactérias oxidadoras de alcano, sob condições controladas em casa de vegetação, utilizando técnicas moleculares para quantificar (qPCR), análisar a estrutura (T-RFLP) e a diversidade da comunidade (sequenciamento de metagenoma). Para isso o solos coletado de áreas de plantação de cana-de-açúcar com os manejos de colheita com e sem queima da palhada, foram utilizados em experimento de mesocosmos composto por vasos com planta, de onde foi coletado solo rizosférico, e vaso sem planta, de onde foi coletado solo controle. A quantificação dos gene 16S rRNA de Actinobacteria e do gene alkB não mostraram diferenças estatística nas comparações dos solos controles de cana-de-açúcar com e sem queima, e na comparação entre os solos rizosférico e controle de cada tipo de manejo de colheita. A análise dos Fragmentos Terminais de Restrição (TRFs) dos solos controle dos diferentes sistemas de colheita, mostraram uma separação da comunidade bacteriana presente no solo, o mesmo ocorreu nas comparações entre os solos rizosférico e controle de diferentes manejos de colheita. Porém, quando se observa a estrutura taxonômica da comunidade bacteriana nota-se que os filos Proteobacteria, Actinobacteria, Firmicutes e Acidobacteria são predominantes nos diferentes solos amostrados, as diferenças estatísticas entre os solos controle e rizosférico de cada tratamento se apresentam a partir da análises das famílias bacterianas presentes nos solos. Para o gene alkB, as sequências encontratas nos diferentes solos pertencem aos dois filos mais abundantes nos solos, os filo Proteobacteria e Actinobacteria, porém algumas sequências pertencem a bacterias não identificadas. Assim, conclui-se que as mudanças na estrutura da comunidade bacteriana presente em solo com monocultura da cana-de-açúcar são perceptíveis a partr da análises de grupos filogenéticos mais baixo e que a comunidade de bactéria degradadoras de alcanos são pertencentes, principalmente, os filos Actinobacteria e Proteobacteria, porém ainda é necessário mais estudos para a classificação das bactérias não identificadas / The phylum Actinobacteria is currently considered one of the major phyla belonging to the domain Bacteria. This phylum can represent up to 30% of the soil microbial community. The organisms of the phylum Actinobacteria has been characterized as antibiotic producers and degraders of complex substances such as alkanes, present in several substances found in plants. Regarding the degradation of alkanes it has already been proven that alkB gene (alkane hydroxylase), present in the phylum Actinobacteria, is related to its degradation. But the communities of such organisms can be altered by changes in land use, which in Brazil occur extensively due to implementation of monocultures. The cultivation of sugarcane, which has been expanded annually, appears as one of the crops that can alter the soil microbiota. The different sugarcane harvest systems, with and without straw burning, can alter the soil microbiota.Thus, considering the role of the phylum Actinobacteria in the cycling of organic matter, in addition to their biotechnological importance, this study evaluated how different harvest systems of sugarcane influence rhizospheric communities of Actinobacteria and alkane-oxidizing bacteria under controlled conditions in a greenhouse, using molecular techniques to quantify (qPCR), analyse the structure (T-RFLP) and diversity of the community (metagenomic sequencing). For this, the soil collected from sugarcane fields with harvest managements with and without burning the straw, was used in a mesocosms experiment composed of pots with a sugarcane plant, from where rhizosphere soil was collected, and without plant, from where control soil was collected. The quantification of Actinobacteria 16S rRNA gene and alkB gene showed no statistical differences in the comparison of sugarcane controls soil with and without burning, and in the comparison between rhizosphere and control soil of each type of crop management. Terminal Restriction Fragments (TRF) analysis of control soils of different harvesting systems showed a separation of the bacterial community present in the soil, the same occurred in the comparison between rhizosphere and control soils of different harvesting systems. However, when observing the taxonomic structure of the bacterial community it is noted that that the phyla Proteobacteria, Actinobacteria, Firmicutes and Acidobacteria are predominant in different sampled soils, the statistical differences between the control and rhizosphere soil of each treatment are presented from the analysis of bacterial families present in the soil. For alkB gene, the sequences found in different soils belong to the two most abundant phyla in the soil, the phylum Proteobacteria and Actinobacteria, but some sequences belong to unidentified bacteria.Thus, it is concluded that the changes in the structure of the bacterial community present in soil with monoculture of sugarcane are apparent from the analysis of lower phylogenetic groups and the alkane-degrading bacterial communities belong mainly to the phyla Actinobacteria and Proteobacteria, however it is still needed further studies for the classification of unidentified bacteria

O filo Actinobacteria é, atualmente, considerado um dos maiores filos pertencentes ao domínio Bacteria. Este filo pode representar até 30% da comunidade microbiana do solo. Os organismos do filo Actinobacteria tem sido caracterizados como produtores de antibiótico e degradadores de substâncias complexas como os alcanos, presente em diversas substâncias encontradas em plantas. Em relação à degradação dos alcanos já tem sido comprovado que o gene alkB (alcano hidroxilase), presente no filo Actinobacteria, está relacionado a sua degradação. Porém, as comunidades de tais organismos podem ser alteradas por mudanças no uso do solo, que no Brasil ocorrem extensivamente devido a implantação de monoculturas. A cultura de cana-de-açúcar, que vem se expandido anualmente, se mostra como uma das culturas que podem alterar a microbiota do solo. Os diferentes sistemas de colheita de cana-de-açúcar, com e sem a queima da palhada, podem alterar a microbiota do solo. Assim, considerando o papel do filo Actinobacteria na ciclagem de matéria orgânica, além da sua importância biotecnológica, este trabalho avaliou como os diferentes sistemas de colheita da cana-de-açúcar influenciam as comunidades rizosféricas de Actinobacteria e de bactérias oxidadoras de alcano, sob condições controladas em casa de vegetação, utilizando técnicas moleculares para quantificar (qPCR), análisar a estrutura (T-RFLP) e a diversidade da comunidade (sequenciamento de metagenoma). Para isso o solos coletado de áreas de plantação de cana-de-açúcar com os manejos de colheita com e sem queima da palhada, foram utilizados em experimento de mesocosmos composto por vasos com planta, de onde foi coletado solo rizosférico, e vaso sem planta, de onde foi coletado solo controle. A quantificação dos gene 16S rRNA de Actinobacteria e do gene alkB não mostraram diferenças estatística nas comparações dos solos controles de cana-de-açúcar com e sem queima, e na comparação entre os solos rizosférico e controle de cada tipo de manejo de colheita. A análise dos Fragmentos Terminais de Restrição (TRFs) dos solos controle dos diferentes sistemas de colheita, mostraram uma separação da comunidade bacteriana presente no solo, o mesmo ocorreu nas comparações entre os solos rizosférico e controle de diferentes manejos de colheita. Porém, quando se observa a estrutura taxonômica da comunidade bacteriana nota-se que os filos Proteobacteria, Actinobacteria, Firmicutes e Acidobacteria são predominantes nos diferentes solos amostrados, as diferenças estatísticas entre os solos controle e rizosférico de cada tratamento se apresentam a partir da análises das famílias bacterianas presentes nos solos. Para o gene alkB, as sequências encontratas nos diferentes solos pertencem aos dois filos mais abundantes nos solos, os filo Proteobacteria e Actinobacteria, porém algumas sequências pertencem a bacterias não identificadas. Assim, conclui-se que as mudanças na estrutura da comunidade bacteriana presente em solo com monocultura da cana-de-açúcar são perceptíveis a partr da análises de grupos filogenéticos mais baixo e que a comunidade de bactéria degradadoras de alcanos são pertencentes, principalmente, os filos Actinobacteria e Proteobacteria, porém ainda é necessário mais estudos para a classificação das bactérias não identificadas / The phylum Actinobacteria is currently considered one of the major phyla belonging to the domain Bacteria. This phylum can represent up to 30% of the soil microbial community. The organisms of the phylum Actinobacteria has been characterized as antibiotic producers and degraders of complex substances such as alkanes, present in several substances found in plants. Regarding the degradation of alkanes it has already been proven that alkB gene (alkane hydroxylase), present in the phylum Actinobacteria, is related to its degradation. But the communities of such organisms can be altered by changes in land use, which in Brazil occur extensively due to implementation of monocultures. The cultivation of sugarcane, which has been expanded annually, appears as one of the crops that can alter the soil microbiota. The different sugarcane harvest systems, with and without straw burning, can alter the soil microbiota.Thus, considering the role of the phylum Actinobacteria in the cycling of organic matter, in addition to their biotechnological importance, this study evaluated how different harvest systems of sugarcane influence rhizospheric communities of Actinobacteria and alkane-oxidizing bacteria under controlled conditions in a greenhouse, using molecular techniques to quantify (qPCR), analyse the structure (T-RFLP) and diversity of the community (metagenomic sequencing). For this, the soil collected from sugarcane fields with harvest managements with and without burning the straw, was used in a mesocosms experiment composed of pots with a sugarcane plant, from where rhizosphere soil was collected, and without plant, from where control soil was collected. The quantification of Actinobacteria 16S rRNA gene and alkB gene showed no statistical differences in the comparison of sugarcane controls soil with and without burning, and in the comparison between rhizosphere and control soil of each type of crop management. Terminal Restriction Fragments (TRF) analysis of control soils of different harvesting systems showed a separation of the bacterial community present in the soil, the same occurred in the comparison between rhizosphere and control soils of different harvesting systems. However, when observing the taxonomic structure of the bacterial community it is noted that that the phyla Proteobacteria, Actinobacteria, Firmicutes and Acidobacteria are predominant in different sampled soils, the statistical differences between the control and rhizosphere soil of each treatment are presented from the analysis of bacterial families present in the soil. For alkB gene, the sequences found in different soils belong to the two most abundant phyla in the soil, the phylum Proteobacteria and Actinobacteria, but some sequences belong to unidentified bacteria.Thus, it is concluded that the changes in the structure of the bacterial community present in soil with monoculture of sugarcane are apparent from the analysis of lower phylogenetic groups and the alkane-degrading bacterial communities belong mainly to the phyla Actinobacteria and Proteobacteria, however it is still needed further studies for the classification of unidentified bacteria

Made available in DSpace on 2014-12-17T15:18:12Z (GMT). No. of bitstreams: 1 MatheusSP.pdf: 232848 bytes, checksum: 1ddb7fd054909bb2524f84dc7c2b0a7f (MD5) Previous issue date: 2007-02-28 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / Some microorganisms from virgin ecosystems are able to use petroleum it as source of carbon and energy. The knowledge of microbial biodiversity can help to reveal new metabolic systems for utilization alkanes with biotechnological importance. The aim of this study is: i) Accomplish an in silico study of the AlkB protein aimed to understand the probable mechanism involved on selectivity of alkanes in Gram positive and Gram negative bact?ria. ii) prospect and analyze the response of the microbial alkanotrophics communities in soil and mangrove sediments of BPP RN and soil of Atlantic forest in the Horto Dois Irm?os Reserve area/PE using the molecular biomarker, gene alkB; with the PCR and PCR-DGGE approach / Alguns microrganismos de ecossistemas virgens s?o capazes de utilizar petr?leo como fonte de carbono e energia. O conhecimento dessa biodiversidade microbiana pode revelar vias metab?licas microbianas para utilizar alcanos que venham a ter import?ncia biotecnol?gica. Os objetivos do presente trabalho s?o: i) realizar estudos in silico da prote?na AlkB visando obter subs?dios para a compreens?o do mecanismo de seletividade na utiliza??o de alcanos em bact?rias Gram positivas e Gram negativas; ii) prospectar e avaliar a resposta de comunidades microbianas alcanotr?ficas de solos e mangue da BPP RN e solo da Mata Atl?ntica na Reserva Horto Dois Irm?os-PE utilizando o gene alkB como biomarcador molecular, e as t?cnicas de PCR e PCR-DGGE

Regulation of gene expression in response to cellular and organismal needs is essential for sustaining organisms' survival and successful competition in the evolution of life forms. This regulation is executed at multiple levels starting with regulation of gene transcription, followed by regulation at multiple posttranscriptional levels. In this thesis, I focused on posttranscriptional mechanisms that contribute to gene expression regulation in the model organism Caenorhabditis elegans which enables powerful genetic and genomic techniques and allows the visualization of experimental genetic manipulations in toto, on the level of the complete organism during its life span. For this, we analysed the function of the orthologue of mammalian transcriptional corepressor NCOR, GEI-8. We used a functionally defective mutant gei-8(ok1671). I analysed the whole genome expression of homozygous gei- 8(ok1671) mutant and its link with observed mutant phenotype that includes defective gonad development and sterility and performed experiments leading to the proposition that disbalances in 21-U RNAs of piRNA class present in the most derepressed gene, the predicted mitochondrial sulfide:quinine reductase encoded by Y9C9A.16, are associated with the gonadal phenotype. In the second part of the thesis, I focused on...

This work discusses applications of computational simulations to enzymatic systems with a particular focus on the effects of various small perturbations on cancer and disease-related systems. First, we cover the development of carbohydrate-based PET imaging ligands for Galectin-3, which is a protein overexpressed in pancreatic cancer tumors. We uncover several structural features for the ligands that can be used to improve their binding and efficacy. Second, we discuss the AlkB family of enzymes. AlkB is the E. coli DNA repair protein for alkylation damage, and has human homologues with slightly different functions and substrates. Each has a conserved active site with a catalytic iron and a coordinating His...His...Asp triad. We have applied molecular dynamics (MD) to investigate the effect of a novel single nucleotide polymorphism for AlkBH7, which is correlated with prostate cancer and has an unknown function. We show that the mutation leads to active site distortion, which has been confirmed by experiments. Thirdly, we investigate the unfolding of hen egg white lysozyme in 90% ethanol solution and low pH, to show the initial steps of unfolding from a native-like state to the disease-associated beta-sheet structure. We compare to mass spectrometry experiments and also show differing pathways based on protonation state. Finally, we discuss three different DNA polymerase systems. DNA polymerases are the primary proteins that replicate DNA during cell division, and have various extra or specific functions. We look at a proofreading-deficient DNA polymerase III mutant, the effects of solvent on DNA polymerase IV's ability to bypass bulky DNA adducts, and a variety of mutations on DNA polymerase kappa.

Genomic integrity is important for living cells' correct functioning and propagation. Deoxyribonucleic acid as a molecule is a subject to chemical reactions with agents that can come from environment as well as from internal metabolism processes. These reactions can induce damage to DNA and thus compromise the genetic information, and result in disease and death of an organism. To mitigate the damage to DNA, cells have evolved to have multiple DNA repair pathways. Presented here is a computational study of DNA repair genes. The structure of the Homo sapiens direct DNA repair gene ALKBH1 is predicted utilizing homology modeling methods and using AlkB and DBL proteins as templates. Analysis of the obtained structure and molecular dynamics simulations give insights into potentially functionally important residues of the protein. In particular, zinc finger domains are predicted, and lysines that could perform catalytic activities are investigated. Subsequent mutagenesis experiments revealed the effect of the residues predicted to form zinc fingers on activity of ALKBH1. Structure and dynamics of AlkD, a Bascillus cereus base excision DNA repair protein is also studied. This protein has been shown to bind DNA with large alkyl adducts and perform excision catalysis without base flipping which is characteristic to other enzymes in the same family. MD simulations of AlkD revealed that B helix, which interacts with DNA, has higher fluctuations when AlkD is not bound to DNA, and thus could have a role in binding and recognition of DNA. For the purpose of finding biomarkers and to further our understanding of a mode of action of DNA repair genes, statistical methods were applied to identify mutations that are linked to cancer phenotypes. Analysis was based on case-control studies of patients with cancers of prostate, breast, pancreas, lung as well as chronic lymphocytic leukemia from NCBI dbGAP database. Those mutations that result in missense mutations were further investigated. In particular, extensive MD simulations and experimental investigations were performed on the mutation in the ALKBH7 gene that was found to be linked to prostate cancer.