INTRODUÇÃO: As Aeromonas são bactérias distribuídas predominantemente em meio aquático. São consideradas patógeno emergente, podendo causar doenças em peixes como também no homem. Os problemas mais comuns são a gastrenterite no homem e morte em peixes. OBJETIVO: Este estudo foi desenvolvido para comparar a identificação fenotípica com a genotípica, e também para conhecer o perfil de resistência aos antibióticos em Aeromonas caviae, A. aquariorum, e A. sanarellii isoladas do ambiente aquático e a presença de genes de virulência e resistência. MATERIAL E MÉTODOS: O DNA das 24 cepas em estudo foi extraído por choque térmico e purificado utilizando CTAB. Foram realizadas as PCRs para a detecção dos genes de virulência e dos genes de resistência, após a realização do antibiograma. RESULTADOS: Foram identificadas 4 A. caviae das quais 3(75,0 por cento) apresentaram pelo menos um dos genes act, ast ou alt. Das 3 A. aquariorum, 1(33,3 por cento) apresentaram positividade para os genes act e ast. Entre os 5 isolados de A. sanarellii 1(50,0 por cento) possuíam os genes alt e ast. Seis isolados não foram posicionados taxonomicamente entre as espécies descritas de Aeromonas, e dentre essas um exemplar apresentou o gene alt. Em relação às enzimas MBL e AmpC foram obtidos respectivamente: 3(100 por cento) e 3(100 por cento) em A. aquariorum; 2(50,0 por cento) e 4(100 por cento) em A. caviae; 3(75,0 por cento) e 5(100 por cento) em Aeromonas spp.; 1(20 por cento) e 5(100 por cento) A. sanarellii; Nenhum isolado apresentou resultado positivo, no teste fenotípico, para a produção de ESBL. Com a realização da PCR foi detectada a presença de 5 amostras com gene tipo blaMOX, 21blaCPHA , 17 tipo blaTEM e 2 cepH. CONCLUSÃO: Os resultados sugerem que os isolados podem servir potencialmente como reservatórios de resistência aos antimicrobianos e ainda, que os isolados podem ser considerados patógeno emergentes e significativos para a saúde pública / INTRODUCTION: Aeromonas spp. is predominantly distributed in the aquatic environment. They are regarded as emerging pathogen, causing disease in fish but also in man. The most common problems are gastroenteritis in humans and death in fish. OBJECTIVE: This study was designed to compare phenotypic with genotypic identification, and also to know the profile of antibiotic resistance in Aeromonas caviae, A. aquariorum, and A. sanarellii isolated from aquatic environment and the presence of virulence genes and resistance. MATERIAL AND METHODS: DNA from 24 strains under study was extracted by thermal shock and purified using CTAB. PCR reactions were performed for the detection of virulence and resistance genes, after the completion of the antibiotic resistance test. RESULTS: We identified four A. caviae from which 3(75.0per cent) had at least one of the genes act, ast or alt. From 3 A. aquariorum, 1(33.3per cent) was positive for the genes act and ast. Among the five isolated A. sanarellii, 1(50.0per cent) had the alt and ast genes Six isolates were not positioned within taxonomically described species of Aeromonas, and among these only one strain presented the alt gene. Regarding the MBL and AmpC it was obtained respectively: 3(100per cent) and 3(100per cent) isolates from A. aquariorum; 2(50.0per cent) and 4(100per cent) isolates from A. caviae; 4(66.7per cent) and 3(50.0per cent) isolates from Aeromonas spp.; and 1(20per cent) and 5 (100per cent) isolates from A. sanarellii. None of the isolates showed positive results in the phenotypic test for ESBL production. The PCR reaction detected the presence of 5 strains with blaMOX-like gene; 21 with blaCPHA gene; 17 with blaTEM-like gene and 2 with cepH gene. CONCLUSION: These findings suggest that the isolates may serve as potential reservoirs of antimicrobial resistance and also that the isolates could be considered emerging pathogens of public health significance

As toxinas produzidas por cianobactérias em ecossistemas aquáticos de superfície utilizados para abastecimento público constituem uma preocupação mundial, com casos de intoxicação relatados em diversos países.Sérios problemas de saúde e até mesmo óbito podem ocorrer como consequência dessas intoxicações. Em ambientes de água doce eutrofizados, florações de espécies de Microcystis são frequentemente observadas e, devido à sua ampla distribuição geográfica e capacidade de produzir toxinas, este é um dos gêneros de cianobactérias mais extensivamente estudados. Microcystis spp. são conhecidas pela produção da potente hepatotoxina microcistina. No entanto, um estudo recente com a linhagem M. aeruginosa SPC777 isolada da represa Billings (São Paulo, SP) relatou a sua capacidade de produção simultânea da [L-ser7] microcistina-RR e da neurotoxina saxitoxina (goniautoxinas 1, 2, 3 e 4). Esse foi o primeiro relato de produção de saxitoxina por uma cianobactéria unicelular. Nesse contexto, o presente estudo teve como objetivo identificar e sequenciar os genes envolvidos na biossíntese da microcistina e saxitoxina na linhagem M. aeruginosa SPC777 e reavaliar a produção destas toxinas após vários anos de cultivo em laboratório. Para isso, foi feito o sequenciamento do genoma de M. aeruginosa SPC777 na plataforma SOLiD V3 e a montagem ab initio das leituras foi realizada usando os algoritmos Edena e Velvet. Análises Blast no banco de dados do NCBI foram realizadas na busca de similaridade por sequências dos genes mcy e sxt e de genes que flanqueiam os agrupamentos envolvidos na biossíntese de ambas as toxinas. Além disso, PCR e sequenciamento Sanger foram empregados para auxiliar a busca dos genes de interesse. Os dez genes envolvidos na biossíntese da microcistina (mcyA-J) foram encontrados e anotados a partir do genoma da M. aeruginosa SPC777, assim como os genes dnaN e uma1, que são normalmente encontrados flanqueando o agrupamento gênico da microcistina. O arranjo dos genes mcy no agrupamento seguiu a mesma ordem de outros descritos na literatura, mas foram encontradas diferenças na sequência de nucleotídeos para alguns dos genes. Para saxitoxina, apenas cinco genes entre aqueles diretamente envolvidos na biossíntese desta neurotoxina foram encontrados usando PCR e sequenciamento Sanger. As sequências parciais dos genes sxt apresentaram alta identidade com outros encontrados em cianobactérias tóxicas. Além disso, a tradução dessas sequências em aminoácidos e as funções protéicas e domínios preditos confirmaram sua identidade como genes da sintetase de saxitoxina. Análises químicas em HPLC-MS/MS mostraram a produção de microcistina, com a detecção do íon m/z 1036, que corresponde à microcistina-YM. Entretanto, não foi observada produção de saxitoxinas. Pelo que sabemos, este é o primeiro agrupamento gênico de microcistina sequenciado de uma linhagem de Microcystis isolada da América do Sul,além de serem os primeiros genes sxt descritos em uma cianobactéria unicelular. Este estudo propiciou novos conhecimentos sobre a origem dos genes mcy e sxt e contribuiu para uma melhor compreensão da evolução destas toxinas / The toxins produced by cyanobacteria in surface aquatic ecosystems used for public supply constitute a worldwide concern, with poisoning cases reported in several countries. Serious health problems and even death can occur as a consequence of these poisonings. In eutrophic freshwater environments, blooms of Microcystis species are often observed and, due to its wide geographic distribution and ability to produce toxins, this is one of the most extensively studied cyanobacterial genera. Microcystis spp. are known for the production of the potent hepatotoxin microcystin. Nonetheless, a recent study with the strain M. aeruginosa SPC777 isolated from Billings reservoir (São Paulo, SP) reported its ability for simultaneous production of [L-ser7] microcystin-RR and the neurotoxin saxitoxin (gonyautoxins 1, 2, 3 and 4). This was the first report of saxitoxin production by a unicellular cyanobacterium. In this context, the present study aimed at the identification and sequencing of the genes involved in the biosynthesis of microcystin and saxitoxin in the strain M. aeruginosa SPC777 and re-evaluation ofthe production of these toxins after several years of cultivation in laboratory. For this, whole genome sequencing of M. aeruginosa SPC777 was done in the platform SOLiD V3 and ab initio assembly of the reads was performed using the algorithms Edena and Velvet. Blast analyses in the NCBI database were performed in the searchfor similarity to mcy and sxt gene sequences and to genes flanking the clusters involvedin the biosynthesis of both toxins. Furthermore, PCR and Sanger sequencing were employed to help the search for the genes of interest. The ten genes involved in microcystin biosynthesis (mcyA-J) were found and annotated from the genome of M. aeruginosa SPC777, as well as the genes dnaN and uma1, which are usually found flanking the microcystin gene cluster. The arrangement of mcy genes in the cluster has followed the same order than others described in literature, but differences were found in the sequence of nucleotides for some of the genes. For saxitoxin, only five genes among those directly involved in the biosynthesis of this neurotoxin were found using PCR and Sanger sequencing. The partial sxt gene sequences have shown high identities to others found in toxic cyanobacteria. Additionally, their translation into amino acids and the predicted protein functions and domains confirmed their identity as saxitoxin synthetase genes.HPLC-MS/MS chemical analyses have shown the production of microcystin, with the detection of the ion m/z1036, which corresponds to the microcystin-YM. Nevertheless, saxitoxin production was not observed. As far as we know, this is the first microcystin gene cluster sequenced from a Microcystis strain isolated from South America and it is also the first time that sxt genes are described in a unicellular cyanobacterium. This study has brought new insightson the origin of the mcy and sxt genes and contributed to a better understanding of the evolution of these toxins

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"January 2003" / Addendum on back page. / Bibliography: p. 175-184. / 184 p. : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / "In the study reported in this thesis, a PCR-based approach was used to isolate the b-like globin genes that are present in the tammar wallaby, Macropus eugenii, including the gene that encodes the w-globin chain. Three -like globin genes (b-, e-, w-) that had previously been described at the protein level in the tammar wallaby were characterised. w-globin orthologues were also identified in a wide range of marsupial species, and in one of these species, the dunnart (Sminthopsis crassicaudata), the complete DNA sequence of the w-globin gene was determined. Southern analysis in the dunnart and in situ hybridisation in the tammar wallaby, provided evidence for the unexpected conclusion that w-globin is not part of the -globin gene cluster in these species. RT-PCR studies using RNA isolated from a new-born dunnarts confirmed that w-globin is expressed in this species. Therefore, this is the first report of an "orphaned" b-like globin gene that is expressed in a vertebrate." --p. 6. / Thesis (Ph.D.)--University of Adelaide, Dept. of Molecular Biosciences, 2003

Thesis (M.S.)--University of Missouri--Rolla, 2007. / Vita. The entire thesis text is included in file. Title from title screen of thesis/dissertation PDF file (viewed December 7, 2007) Includes bibliographical references (p. 28-35).

The overall goal of my work has been to gain a better understanding of Axenfeld-Rieger Syndrome (ARS), a human autosomal dominantly inherited mal-development of the anterior segment of the eye that is associated with glaucoma. By studying rare genetic causes of this complex disease we are gaining insight into the initial steps that ultimately lead to blindness. To achieve the goal of better understanding ARS, my research project had two parts. In the first part, I performed a retrospective clinical study in which I analyzed the glaucoma-related clinical presentation of ARS patients with FOXC1 and PITX2 defects. This study showed a good genotype-phenotype correlation which may be important for the physician in dealing with ARS patients. Patients with FOXC1 mutations had the mildest prognosis in glaucoma development, while patients with PITX2 defects and patients with FOXC1 duplication had a more severe prognosis in glaucoma development than patients with FOXC1 mutations. I tried to determine the best treatment for glaucoma in these patients. Unfortunately, in this study, current medical therapies did not successfully lower intraocular pressure or prevent progression of glaucoma in ARS patients with FOXC1 or PITX2 alterations. This clinical study also provided useful diagnostic criteria to identify the gene responsible for ARS. The second part of the project was to study the gene regulatory pathways of the PITX2 gene, mutations of which cause ARS. PITX2 is a transcription factor that regulates the expression of genes in the eye. The discovery of direct downstream targets of PITX2 is necessary for understanding the genetic mechanisms underlying complex, highly regulated processes such as development and underlying heritable human disorders. To find direct target genes of PITX2, I have used a recently developed method: the hormone receptor (HR)-inducible expression system for transcription factors coupled microarray analysis. The results obtained using this method have involved PITX2 in control of cellular stress. Recent investigations have suggested significant roles for cellular stress in glaucoma pathology. Understanding the control of these key aspects of cell function will have profound implications for understanding and treating the glaucoma that is the most clinically serious consequence of mutations of PITX2.

Thesis (Ph. D.)--Ohio State University, 2006. / Available online via OhioLINK's ETD Center; full text release delayed at author's request until 2007 Jun 15

M.S. / Molecular Biology / This thesis describes the cloning and analysis of PER6, a gene required for peroxisome biogenesis in Pichia pastoris. The gene was cloned by functional complementation of a per6 P. pastoris mutant strain that was one of a number of peroxisome-deficient mutants isolated in this laboratory. The complementing activity was localized to a small DNA fragment by subcloning and Northern filter hybridization analysis and the DNA sequence of the fragment was determined. The sequence revealed a 1296-bp open reading frame which potentially encodes a 432-amino acid protein of 49 kD. The gene was transcribed into a message of 1.4 kilobases that was constitutively expressed but induced several-fold in cells growing on methanol. A mutant strain with a deletion of a large portion of the open reading frame was constructed and used to genetically demonstrate that the cloned gene was identical to the defective gene in the originally isolated per6 mutant. The predicted amino acid sequence of the PER6 product revealed several interesting features, including a significant regional similarity to PAF-1, a gene known to be defective in some patients with Zellweger syndrome, a lethal human genetic disease caused by peroxisome deficiency. Finally, the PER6 product was produced in E. coli and purified to serve as antigen for antibody production.

Antibiotics are frequently used in aquaculture for the treatment of bacterial diseases. In this study, bacteria were isolated from the spleen, liver and kidney of grouper from PCG and cobia from EMD and THOD in southern Taiwan. All isolates were cultured on TCBS agar, blood agar, and MacConkey agar. The results showed that the isolates from PCG were 83% (20/24) Vibrio and 13% (3/24) £]-hemolysis¡F76% (22/29) Vibrio and 24% (7/29) £]-hemolysis from EMD¡F100% (6/6) Vibrio and none of £]-hemolysis from THOD. The bacteria were tested for antibiotics resistance by the disc agar diffusion method. 70% bacteria resisted to penicillin, cephazolin, and streptomycin while double resistance to furazolidone/streptomycin increased to with time from 0% to 83% in PCG. In EMD, 70% bacteria resisted to streptomycin and furazolidone, and double resistance to furazolidone/streptomycin increased with time from 0% to 60%. In THOD, 50% bacteria resisted to £]-lactam drugs, 100% bacteria resisted to cephazolin, and 67% bacteria doubly resisted to ampicillin/amoxycillin, cephalexin/cephazolin, cephalexin/streptomycin and cephazolin/streptomycin. Further, the detection of tetB, tetD, tetM, tetS and tetX resistance, tetracycline resistance genes, in the chromosomal DNAs from 17 multiple resistance isolates were performed by PCR. The PCR products were confirmed by digestion of restriction enzymes. The data indicated that J39-1, J39-2, K26-4 and K27-2, strains from THOD, together with N18-5 and M35-2, from PCG, were identified as carrying tetB. From EMD, The tetB and tetM genes were detected in P19-1, P19-3, P32-1 and Q8-3, whereas strain O2-3 carried tetS gene.