Orientadores: Reginaldo Bruno Gonçalves, Daniel Saito / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-16T03:29:19Z (GMT). No. of bitstreams: 1 Cruz_SergioEduardoBragada_D.pdf: 4484196 bytes, checksum: 41cbfe2b81d194ae03d4070bbe8108b8 (MD5) Previous issue date: 2010 / Resumo: Há um consenso que outros micro-organismos além de Aggregatibacter actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), Tannerella forsythia (Tf) e Treponema denticola (Td) estariam correlacionados às periodontites, inclusive algumas espécies ainda não identificadas. Nosso objetivo foi estudar as microbiotas subgengivais de indivíduos com periodontite crônica generalizada (PCG) e periodontite agressiva generalizada (PAG) para avaliar diferenças entre suas microbiotas. MATERIAL E MÉTODOS-Foram selecionados 15 indivíduos com PCG e 14 com PAG. Coletou-se amostra do biofilme subgengival de uma bolsa periodontal profunda (BP-PS ? 7mm) e uma moderada (BM - PS entre 5 e 6 mm) de cada indivíduo. Foi preparado e analisado, por meio de DGGE, o perfil bacteriano entre os grupos. A similaridade e a análise de cluster do padrão de UTO's foram verificadas utilizando-se coeficiente de Jaccard e a construção do dendrograma realizada por UPGMA. Realizou-se também análise clonal direta de 10 amostras de BP de cada grupo e as sequências foram agrupadas em táxons com similaridade >97%. RESULTADOS-DGGE - No perfil de DGGE foi observada uma tendência para a formação de grupos em BP, mas não em BM, com a presença de dois grupos maiores e distintos de oito indivíduos tanto para PCG como PAG, com variação de similaridade intra-grupo entre 53,6-68,4% e 50,2-64,7%, respectivamente. Análise clonal - Foram identificados 109 táxons conhecidos a partir de 987 clones. Ao todo 44 gêneros bacterianos, 28 gêneros comuns aos dois grupos, nove que se apresentaram apenas para PCG e sete para PAG. Entre os dois grupos foram observados 34 táxons comuns, sendo 42 específicos para PAG e 37 para PCG. A espécie Tf foi detectada em 90% dos indivíduos com PCG e 80% com PAG, Pg foi detectada em 70% com PCG e 50% com PAG e Td foi detectada em 40% com PCG e 30% PAG. A espécie Aa foi encontrada em somente 20% de PCG e 30% de PAG. A espécie Filifactor alocis foi observada em altas taxas e prevalência em PCG (58 clones, 90%) e PAG (91 clones, 90%). As espécies encontradas exclusivamente por grupo com prevalência acima de dois pacientes foram: PCG: Treponema lecithinolyticum, Selenomonas dianae, Prevotella pleuritidis, Dialister pneumosintes; e para PAG: Fusobacterium nucleatum ss vincentii, Veillonella parvula, Peptococcus sp. Cepa GEA8, Streptococcus gordonii, Lautropia mirabilis, Gemella sanguinis, Afipia broomeae. Para os filotipos, PCG: Peptostreptococcaceae sp. Clone-MCE10_174, Fusobacterium sp. C-I035, Veillonellaceae sp. C-JS031; PAG: Peptostreptococcaceae sp. C-PUS9170, Treponema sp. CG093. Apesar de não haver exclusividade entre grupos, é de nota os filotipos Synergistes sp. clone W028 (80% e 60%) e o clone D084 (70% e 10%) em PCG e PAG, respectivamente. O filotipo Bacteroidetes sp. AU126 foi encontrado tanto em PCG (60%) como PAG (30%). CONCLUSÃO - O presente trabalho demonstrou por meio de DGGE uma tendência a um perfil microbiano comum entre a maioria das amostras estudadas, entretanto, sem seu completo delineamento como dois grupos distintos microbiologicamente. A análise clonal, apesar de algumas espécies específicas entre grupos, demonstrou pequenas diferenças, sem, entretanto, delinear grupos microbiologicamente específicos. / Abstract: There is an agreement that not only the already known periodontopathogens, Aggregatibacter actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), Tannerella forsythia (Tf) and Treponema denticola (Td) would be involved in periodontitis, but also some others micro-organisms not-yet-identified. The scope of this study is to compare the subgingival microbiota in generalized chronic periodontitis (GCP) or generalized aggressive periodontitis (GAP) subjects. MATERIAL AND METHODS - 15 subjects with GCP and 14 with GAP were enrolled. One subgingival biofilm sample from a periodontal deep pocket (DP) with PD ? 7mm and one from a moderate pocket (MP) with PD from 5 to 6 mm were harvested from each subject. The microbial profiles (OTU's) were compared between groups by DGGE and the similarity OTU profile was analyzed by Jaccard coefficient and the dendrogram and cluster analyses were made by UPGMA. The direct clonal analysis of the 16SrDNA from 10 samples of each group from DP was made. The sequences were grouped in clusters of taxa with > 97% similarity. RESULTS - DGGE - It was observed in the profile a tendency for eight subjects from each group to assemble as clusters in the DP, but not for the MP samples, with similarities between 53.6-68.4% (GCP) and 50.2-64.7% (GAP). Clonal analyses - One-hundred-and-nine already recognized taxa were obtained from 987 clones. From a total of 44 bacterial genera, 28 were common for both groups; nine were exclusive to PCG and seven to PAG subjects. It was found 34 common taxa between GCP and GAP, 37 were specific for GCP and 42 for GAP. The Tf species was found in 90% from GCP subjects and 80% from GAP subjects, Pg was found in 70% from GCP and in 50% from GAP and Td was detected in 40% from GCP and 30% from GAP. The Aa species were found in only 20% GCP subjects and in 30% from GAP. Filifactor alocis species were detected in high prevalence in both GCP (58 clones, 90%) and PAG (91 clones, 90%). The species which were detected exclusively in each group, with 20% prevalence or more were, for GCP: Treponema lecithinolyticum, Selenomonas dianae, Prevotella pleuritidis, Dialister pneumosintes; and GAP: Fusobacterium nucleatum ss vincentii, Veillonella parvula, Peptococcus sp. Cepa GEA8, Streptococcus gordonii, Lautropia mirabilis, Gemella sanguinis, Afipia broomeae. In relation to phylotypes, PCG: Peptostreptococcaceae sp. Clone-MCE10_174, Fusobacterium sp. C-I035, Veillonellaceae sp. C-JS031; PAG: Peptostreptococcaceae sp. C-PUS9170, Treponema sp. C-G093. Despite not been found exclusively for neither GCP nor GAP, the phylotypes Synergistes sp. clone W028 (80% e 60%) and clone D084 (70% e 10%) had a notable presence in GCP and GAP, respectively. The phylotype Bacteroidetes sp. AU126 was found in GCP (60%) and GAP (30%) groups. CONCLUSION - The present study demonstrated by DGGE a slight tendency to the clustering of the microbial profile of some GCP and GAP subjects, although these were not well delineated. The clonal analyses showed some differences, but also could not show GCP and GAP as microbiologic distinct profiles. / Doutorado / Microbiologia e Imunologia / Doutor em Biologia Buco-Dental

No description available.

Masters of Science / Metagenomics has been successfully used to discover novel enzymes from uncultured microorganisms in the environment. In this study, metagenomic DNA from a Malawian hot spring soil sample was used to construct a fosmid library. This metagenomic library comprised of more than 10000 clones with an average insert size of 30 kb, representing more than 3.0 x 108 bp of metagenomic DNA (equivalent to approximately 100 bacterial genomes). The library was screened for cellulase activity using a Congo red plate assay to detect zones of carboxymethylcellulose hydrolysis. This yielded 15 positive fosmid clones, of which five were further characterised for activity and thermostability using the 3, 5-dinitrosalicylic assay. Two of the five fosmids (XP008C2 and XP026G5) were selected for DNA pyrosequencing. The full sequence of the XP008C2 (29800bp) fosmid insert is presented in this study and genes thereon were chosen for further study. / South Africa

Brain tissue samples were collected from individuals with histologically and biochemically confirmed Alzheimer's Disease (AD), as well as from a group of individuals without any signs of neurological disease (NNC). Ribonucleic acid (RNA) was extracted from these tissues, characterized by several chemical methods, and the yields were compared between AD and NNC groups. High molecular weight RNA could be effectively extracted from frozen postmortem human brain. In comparison to the NNC group, tissue RNA levels were reduced in the AD hippocampus, but not in the temporal cortex or substantia innominata (SI). No difference in the physical integrity of the RNA was apparent between AD and NNC groups. A high complexity complementary deoxyribonucleic acid (cDNA) library was prepared using RNA extracted from the NNC SI. Differential hybridization screening using a variety of cDNA probes was employed to identify mRNAs expressed differentially between AD and NNC tissue, and between SI and other human tissues. Many selected mRNAs were examined for specificity of expression in brain tissue and brain regions. The cDNA clone pSI3a-24 identified an mRNA, which, on Northern blot hybridization, was expressed in brain tissue but not in the other human tissues examined. The identified mRNA was unusually large, with a chain length estimated at 15,500 bases. Quantification of the brain tissue levels of this mRNA was carried out using a ribonuclease protection assay. Tissue levels were higher in the SI (40 pg/μg RNA) than in the temporal cortex (28.6 pg/μg), and were lowest in the cerebellum (11.2 pg/μ9). Levels of the mRNA in temporal cortex samples were increased 29% in the AD group, relative to NNC. No significant difference in the SI tissue levels was observed between AD and NNC groups. Hybridization analysis of human genomic DNA indicated that the mRNA was encoded by a single copy gene. Sequence analysis of the full 3 kilobases of cloned cDNA was completed. Computer database searches failed to identify any known nucleic acid sequence with homology to the cDNA. Examination of the cDNA sequence for potential polypeptide coding regions suggested that the corresponding mRNA has a 3' untranslated region of at least 3 kilobases. / Medicine, Faculty of / Graduate

The oprF gene encoding porin protein F from Pseudomonas aeruginosa was cloned onto a cosmid vector into Escherichia coli. Protein F was expressed in large amounts in E. coli and retained its heat- and reduction-modifiable and immunological characteristics. The cloned oprF gene product was purified from E. coli and characterized with respect to pore-forming ability in black lipid bilayers. Small channels, with an average single channel conductance of approximately 0.4 nS, were observed. A similar small channel size was observed for native protein F. The oprF sequences were used as a DNA-DNA hybridization probe with chromosomal DNA from the 17 IATS (International Antigen Typing Scheme) strains of P. aeruginosa, 52 clinical isolates and the non-aeruginosa Pseudomonads. Conservation of oprF sequences was observed among all the P. aeruginosa strains and to a lesser extent among the non-aeruginosa strains of the P. fluorescens rRNA homology group. Insertion mutations in the oprF gene were created in vivo by Tn1mutagenesis of the cloned gene in E. coli and in vitro by insertion of the streptomycin-encoding Ω fragment into the cloned gene, followed by transfer of the mutated protein F gene back into P. aeruginosa and homologous recombination with the chromosome. The oprF mutants were characterized by gel electrophoresis and immunoblotting, and it was shown that the mutants had lost protein F. The P. aeruginosa oprF mutants were characterized with respect to growth rates, antibiotic permeability and cell surface hydrophobicity. The results of these studies indicated that major alterations in the cell surface had occurred and that the cells were unable to grow in a non-defined liquid medium without added electrolytes. Marginal differences were observed in MICs (minimum inhibitory concentrations) of hydrophilic antibiotics for the oprF mutants compared with their protein F-sufficient parents. The putative roles of protein F in antibiotic permeability and general outer membrane permeability are discussed. Evidence for extensive homologies between protein F, the OmpA protein of E. coli and PHIII of Neisseria gonorrhoeae are presented. A role for protein F in prophylactic anti-Pseudomonas therapy, as a target for vaccine development, is proposed. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

The β-glucosidase (Abg) from ATCC 21400, an Agrobacterium species, was purified to homogeneity. The protein was cleaved with cyanogen bromide and the peptides were purified by reversed phase high pressure liquid chromatography. The partial amino-acid sequences for three CNBr peptides, CNBr1, CNBr2 and CNBr3, were determined by automated Edman degradation. A sequence from CNBr2 was used to synthesize a mixture of oligonucleotides which was used as a hybridization probe to identify a recombinant DNA clone carrying the gene for β-glucosidase. A single clone was isolated which expressed an enzymatic activity that hydrolyzed several β-glucosides. The enzymatic activity produced by this clone could be adsorbed by rabbit antiserum raised against the Agrobacterium enzyme. The direction of transcription of the β-glucosidase gene was determined by verifying the DNA sequence 3' to the oligonucleotide probe binding site. After subcloning the gene a high level of expression was obtained in the plasmid vector pUC18 using the lacZ gene promoter. The nucleotide sequence of the 1599 bp insert in pABG5 was determined using the chain terminator method. The start of the protein coding region was determined by aligning the amino terminal sequence of the protein with the predicted amino acid sequence of the cloned gene. The open reading frame was 1387 nucleotides and contained 458 codons. The molecular weight calculated from the deduced amino acid sequence agreed with that observed from both the native and recombinant enzymes. The predicted amino acid composition from the open reading frame matched with that determined for the native β-glucosidase. The stop codon of this coding region was followed by a potential stem loop structure which may be the transcriptional terminator. There was a region of the deduced Abg sequence which had homology to a region from two other β-glucosidase sequences. This region of homology contained a putative active site by analogy with the active site of hen egg white lysozyme. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 1980 / Vita. / Includes bibliographical references. / by James F. Gusella. / Ph. D. / Ph. D. Massachusetts Institute of Technology, Department of Biology

No description available.

In plants, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR, EC 1.1.1.34) is a key enzyme regulating biosynthesis of phytosterols, plant growth regulators, carotenoids, antimicrobial defense compounds, and numerous other isoprenoids. To initiate molecular studies of HMGR in relation to defense responses in plants, we utilized yeast HMGR cDNA sequences to isolate tomato genomic sequences encoding HMGR. The nucleic acid sequence and gene structure was determined. The tomato HMGR gene (HMG2) contains four exons separated by three introns and encodes a polypeptide of 602 amino acid residues (about 64,714 Da). Two membrane-spanning regions are contained in the NH₂-terminus of the HMGR polypeptide. The COOH-terminus shares significant homology with HMGR sequences from different species. Genomic Southern hybridization analyses reveals that tomato contains 3 to 4 HMGR genes. The HMGz2 gene cross-hybridizes to mRNA of about 2.7 kb which is highly induced in tomato cells treated with fungal elicitors and in stems, leaves, or roots stressed by wounding suggesting that the HMGz2 is a defense-related gene in tomato. Hybridization with a gene specific probe indicates that the HMG2 gene is induced specifically during defense responses and is distinct from the gene(s) expressed during fruit development and ripening. / Ph. D.

Superoxide dismutase is an enzyme which scavenges superoxide radicals and is thought to be a longevity determinant, as there exists a positive correlation between superoxide dismutase concentration and maximum life span potential. The cytosolic CuZn superoxide dismutase in D. melanogaster has been purified and sequenced, but the gene has not been cloned. However, when it is available the CuZn SOD gene may be reintroduced into the Drosophila genome via the P-element transformation system so its effects on the life span potential of Drosophila may be studied. This study describes attempts to clone the CuZn SOD gene from D. melanogaster using two mixed sequence oligonucleotide probes. The SI probe corresponds to amino acids 43-48 of the protein sequence and contains 128 different oligonucleotide sequences representing all possible codon combinations predicted from the amino acid sequence. The GT3 probe is targeted to amino acids 90-95 of the protein. In this probe, deoxyguanosine was placed in positions where all four nucleotides may occur to decrease probe heterogeneity. The probes were used to screen D. melanogaster Canton-S and Oregon-R genomic lambda libraries. Three positive clones isolated from the Canton-S library had identical nucleotide sequence in the GT3 probe binding region, and sequencing of the probe binding site revealed that one member of the GT3 probe had formed a 15 bp duplex with the phage DNA. Screening of the Oregon-R library produced four clones which hybridized with both GT3 and S1 probes. When these phage DNA were hybridized to polytene chromosomes by in situ hybridization, none mapped to 68AB on the third chromosome, the location of the CuZn SOD gene. These results suggest that modification of the classical strategy used in this study is necessary, and implications on probe design are discussed. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate