The bacterio-opsin (bop) gene encodes the protein component of the purple membrane (Pum) of the halophilic Archaeum, Halobacterium halobium. We have studied the role of oxygen, DNA supercoiling, and putative regulatory genes (brp and bat) in the induction of bop gene expression. Studies on the wild-type strain NRC-1 showed that the bop gene was induced more than 20-fold at the transcriptional level and the induction was blocked by aeration of cultures. For a Pum overproducer strain S9, the bop gene was transcribed constitutively at a level 10-fold greater than for NRC-1. Similar results were obtained for the divergently transcribed brp gene located upstream of bop. In the absence of a DNA gyrase inhibitor, novobiocin, superhelical density in H. halobium was found to be 50% more negative than in E. coli. Addition of novobiocin at concentrations subinhibitory for growth resulted in a reduction of bop and brp transcription in both strains, and also partially relaxed plasmid DNA supercoiling to the E. coli level. Slightly higher negative supercoiling of plasmids was observed in NRC-1 under microaerobic conditions, whereas, S9 showed slightly higher supercoiling under aerobic conditions. These results support a model where DNA supercoiling mediates the activation of bop gene transcription in response to oxygen limitation. The bop promoter was subsequently cloned in a halobacterial plasmid and introduced into NRC-1, S9, and S9 mutants, which have insertions in brp or a putative oxygen sensor gene, bat, located downstream of brp. The bop promoter on the plasmid showed similar activity as in the chromosomal locus with high transcription level in S9 and no activity in the brp and bat mutants. These results indicate that bop promoter function is not context-dependent and transcriptional activation of bop occurs when the regulatory genes are located in trans. The possible mechanisms for gene regulation mediated by changes in DNA supercoiling include facilitating the interactions between regulators and RNA polymerase, modulating the rate of open complex formation, changing the rate of abortive initiation, or stabilizing unusual DNA structures, which may be favored by 5M salt and high DNA superhelical density found in H. halobium.

In this dissertation, I report my studies on the resistance of Arabidopsis thaliana ecotype Dijon (Di-0) to turnip crinkle carmovirus (TCV), a simple, single-stranded, positive-sense, plant RNA virus. TCV efficiently replicated in protoplasts prepared from Di-0 callus culture, but it did not move long-distance in Di-0 plants (Simon et al., 1992). Cardamine chlorotic fleck virus (CCFV), the carmovirus most closely related to TCV, infected Di-0 plants systemically (Oh et al., 1995). I found that TCV with the coat protein open reading frame (ORF) from CCFV (TCV-CP$\rm\sb{CCFV})$ and TCV with a single base mutation in the initition codon (AUG to ACG) of the coat protein ORF (TCV-CPm) were infectious on ecotypes Columbia (Col-0, TCV-susceptible) and Di-0 (TCV-resistant). These results indicate that TCV coat protein is the viral determinant for resistance of Di-0 to TCV. In addition, my results suggest that the resistance of Di-0 to TCV is specific to TCV and may involve an RNA degradation activity. I also studied the movement of TCV, CCFV, and TCV-CP$\sb{\rm CCFV}$ in Col-0 and Di-0 plants using a whole plant in situ hybridization technique. The results indicate that TCV was restricted to within small areas around the initial infection sites on inoculated leaves of Di-0 plants, whereas TCV (in Col-0), CCFV and TCV-CP$\sb{\rm CCFV})$ (in Col-0 and Di-0) moved long-distance to metabolic sink tissues (young leaves and roots) at different rates. Many plant RNA viruses are associated with small subviral RNAs, including satellite (sat-) RNAs. Small subviral RNAs require a helper virus for replication and movement, and they often intensify or attenuate the symptoms caused by the helper virus. TCV is associated with sat-RNA C, a sat-RNA that normally intensifies the symptoms of the TCV-M isolate (Simon et al., 1988; Li and Simon, 1990). I will report my work on the mechanism of symptom modulation mediated by sat-RNA C in Arabidopsis plants. My results indicate that symptom modulation by sat-RNA C is mediated by the viral coat protein, and a putative interaction between the 3$\sp\prime$ end of sat-RNA C and the N-terminus of coat protein is involved. Symptom attenuation by sat-RNAs is widely believed to be mediated by inhibition of helper virus replication through competition for replication factors (Roossinck et al., 1992). My study shows that inhibition of virus long distance movement is involved in sat-RNA C-mediated symptom attenuation of TCV-CPm (and probably TCV-CP$\rm\sb{CCFV})$ and inhibition of helper virus replication is not important. In addition, symptom attenuation mediated by sat-RNA C is localized and does not involve the major plant defense pathway. A model that explains sat-RNA C-mediated symptom attenuation is proposed.

Escherichia coli O157:H7 is recognized as a major human enteropathogen. Immunotechnology and DNA based methods were developed for quantitative detection of E. coli O157:H7 in ground beef. A direct spectrophotometric immuno-agglutination assay was developed for quantitation of specific Escherichia coli O157 IgG. Optimum conditions of the assay consisted of 1 × 108 cells/ml, 40°C, and 0.005M phosphate buffer containing 0.05% NaCl at pH 7.4. The assay was able to quantitate (13 to 104) μg IgG/ml of anti O157 IgG in crude antiserum and was effectively used with different batches of locally produced antisera. A new cell lysis solution designated TZ (2.0% Triton X-100 in 0.1 M Tris-HCl buffer plus 2.5 mg sodium azide/ml, pH 8.0) was developed. TZ lysis solution was found superior to a variety of cell lysing methods (d.H2O, PCR buffer, SDS, Triton X-100, proteinase K, and lysozyme in combination with proteinase K) and released the greatest yield of E. coli O157:H7 DNA targets prior to PCR. Highest amplification of E. coli O157:H7 SLT-1 and SLT-2 DNA sequences were achieved with the aid of TZ lysis solution and pellet paint. Using 0.01 M phosphate buffered saline pH 6.0 with the aid of differential centrifugation resulted in 57% ± 6 recovery of E. coli O157:H7 from seeded ground beef. The optimization of PCR reaction mixture resulted in a minimum detection level of 100 SLT-1 DNA sequence compared to 200 DNA targets before the optimization. Prior to optimization, the minimum limits for the SLT-2 DNA sequence was 150 DNA targets compared to 20 after complete optimization. The complete optimization of PCR reaction mixture contained 1 mM MgCl2, 1.6 μM each of the two primers, (2.5 and 5 units) of Taq polymerase for SLT-1 and SLT-2 respectively, and 0.2 mM of Deoxynucleotide Triphosphates (dNTPs). The methodologies developed thereby resulted in the detection of 100 cells of E. coli O157:H7 with SLT-1 primer and 50 cells with SLT-2 primer per 10 grams of ground beef after 5.5 hours of pre-enrichment in 30 ml of TSB+ at 37°C.

Mycobacterial species are diverse organisms, classified into tuberculous and nontuberculous mycobacteria (NTM). Mycobacterium tuberculosis has been thoroughly investigated, but pathogenic NTM have not. The identified technology gap for studying potential antibiotic targets across mycobacteria is that there is not a tool developed for efficiently creating bacterial clones containing these genes with a reporter system to evaluate CRISPR interference (CRi) knockdowns. CRi is a quick and simple way to silence genes. In this study, Golden Gate (GG) cloning compatible Lux reporter plasmids were engineered for the efficient cloning of target genes as transcriptional fusions with luxAB, a luminescent reporter, for use with CRi. Additionally, a CRi plasmid was designed with a Giles integration site so that it could be integrated into the mycobacterial genome with the reporter plasmid, but at a different location. Based on current research, it seems that mycobacterial L,D-transpeptidase enzymes (Ldts), involved in peptidoglycan synthesis, are potential targets for the drug class known as β-lactams and should be further explored. Ldt 2 is of particular interest as research indicates that it may be involved in pathogenicity; therefore, GG cloning of M. smegmatis (Msm) Ldt 2 was performed using the designed GG plasmid. Constructing the GG plasmid (pMV306hsp+luxG13, GG pMV) as well as the CRi + Giles integration plasmid (pLJR962 + pML1357, CRi + Giles) was successful; however, the evaluation of the luminescent reporter with CRi knockdown has yet to be performed.

Made available in DSpace on 2014-07-29T15:26:21Z (GMT). No. of bitstreams: 1 Liana Jayme borges.pdf: 1844496 bytes, checksum: 818c982e89c90b277c44959cde90066a (MD5) Previous issue date: 2010-03-19 / Enteral feeding means the nutrition for special purposes, with controlled intake of nutrients. The advantages of its use often become secondary to complications arising from its contamination, which may be associated with infectious complications. The microbial contamination of enteral feeding may occur during all steps being the handling, particularly critical. Considering the importance of enteral feeding as a therapeutic tool in hospitals and the need to guarantee the microbiological quality of the products offered to critical patients, the present work aimed to evaluate the hygienic and sanitary quality of diets and their ingredients and to identify and characterize phenotypic and genotypically, using the antibiogram and pulsed-field gel electrophoresis, strains of Escherichia coli and Staphylococcus aureus obtained from handlers hands and noses, water, module and enteral nutrition from two public hospital in Goiânia, Brazil in order to investigate the probable source of microbological contamination. A total of 80 samples were collected from enteral nutrition and 140 from hands and noses of handlers involved in the diets manufacturing in hospital 1 (H1), between october/2007 and november/2008 and 80 samples from enteral nutrition and 80 from hands and noses of handlers in hospital 2 (H2), between october/2008 and november/2008. From both hospitals were collected 40 samples from water and module. The samples were submitted to microbiological analysis to verify the presence and numbers of pathogenic and indicator microorganisms. E. coli and S. aureus strains were submitted to antibiogram and PFGE. According to antibiogram, all S.aureus isolates (15) from H1 were susceptible to oxacillin, vancomycin, ciprofloxacin and gentamicin. Resistence profile was observed in 10 (66.7%) isolates for penicillin, four (26.7%) isolates for tetracycline and nine (60.0%) isolates for erythromycin, allowing to classify the strains in six different phenotypes (A-F), but it was not efficient for the determination of the bacterial source for the diets. In the H1, all (08) E. coli strains were susceptible to trimethoprim, ciprofloxacin, cephalothin, gentamicin, ceftazidime and tetracycline. Resistence was observed in six (75.0%) isolates for ampicilin. In H2, all strains isolated (12) were susceptible to trimethoprim, ciprofloxacin, gentamicin and ceftazidime and resistence was observed in 11 isolates (91.7%) for cephalothin and 12 (100.0%) for tetracycline and ampicillin, grouping them into five different phenotypes (A-D). Microorganisms showed the same phenotypic profile from handlers and diet samples (phenotypes A and C), suggesting that in these cases, the source of microorganisms for the final product was the food handler. The genotypic typing of S. aureus strains by PFGE generated seven different DNA banding profiles and the E. coli genotyping generated five profiles. Based on the results, two E. coli strains isolated from diets were identical to one strain isolated from food handler from H2 and two of S. aureus isolated from diets were identical to one strain isolated from food handler from H1. This study shows that the enteral feedings showed unsatisfactory sanitary-hygienic conditions in both hospitals and the hand contact is probably one of the sources of greatest significance for enteral diets contamination in the hospital environment. / Entende-se por nutrição enteral a alimentação para fins especiais, com ingestão controlada de nutrientes. As vantagens oferecidas pelo seu emprego muitas vezes tornam-se secundárias às complicações derivadas de sua utilização como a contaminação, que pode estar associada a complicações infecciosas. A contaminação microbiana das fórmulas enterais pode ocorrer em diversas etapas, sendo a manipulação uma etapa especialmente crítica. Tendo em vista a importância da dieta enteral como medida terapêutica em hospitais e a necessidade de se ofertar produtos com qualidade assegurada, devido aos prejuízos que a mesma pode causar aos pacientes, caso esteja contaminada, o objetivo deste estudo foi avaliar a qualidade higiênico-sanitária das dietas e seus ingredientes e caracterizar fenotipicamente, utilizando o antibiograma e, genotipicamente, através da eletroforese em gel em campo pulsado (pulsed-field gel electrophoresis (PFGE), isolados de Escherichia coli e Staphylococcus aureus a partir de manipuladores, água, módulo em pó e dieta enteral de dois hospitais públicos de Goiânia-GO visando estabelecer a possível fonte de microrganismos para o produto final. Um total de 80 amostras de dieta enteral e 140 swabs de mãos e fossas nasais de manipuladores foram coletadas no hospital 1 (H1) entre outubro/2007 e novembro/2008 e 80 amostras de dieta enteral e 80 swabs de mãos e fossas nasais no hospital 2 (H2) entre novembro/2008 e dezembro/2008. Nos dois hospitais foram coletadas também 40 amostras de água e módulo. Foram realizadas análises microbiológicas para contagem de microrganismos indicadores e potencialmente patogênicos. Os isolados de E. coli e S.aureus foram submetidos ao antibiograma e PFGE. De acordo com o antibiograma, todas as cepas de S. aureus isoladas (15) no H1 foram sensíveis à oxacilina, vancomicina, ciprofloxacina e gentamicina. O padrão de resistência foi observado em 10 (66,7%) isolados para penicilina, quatro (26,7%) para tetraciclina e nove (60,0%) para eritromicina, agrupando-os em seis diferentes perfis fenotípicos (A F). Porém, a técnica não foi eficiente em determinar a origem da contaminação das dietas. Para as 20 cepas isoladas de E. coli do H1 e H2, todas (8) do H1 foram sensíveis ao trimetoprim, ciprofloxacina, cefalotina, gentamicina, ceftazidima e tetraciclina. Resistência foi observada em seis (75,0%) isolados para a ampicilina. No H2 todas as cepas isoladas (12) foram sensíveis ao trimetoprim, ciprofloxacina, gentamicina e ceftazidima e resistência foi observado em 11 isolados (91,7%) para a cefalotina e 12 (100,0%) para a tetraciclina e ampicilina, sendo agrupadas em quatro diferentes perfis fenotípicos (A D). Os fenótipos A e C apresentaram microrganismos com o mesmo perfil fenotípico provenientes de manipuladores e dieta, sugerindo que nestes casos, a fonte de microrganismos para o produto final seria os manipuladores. A tipificação genotípica por PFGE originou sete perfis eletroforéticos diferentes para as cepas de S.aureus e cinco para as cepas de E. coli. De acordo com os resultados, duas cepas de E. coli isoladas da dieta foram idênticas a uma cepa isolada do manipulador do H2 e duas cepas de S.aureus isoladas da dieta foram iguais a uma cepa do manipulador do H1. Os dados obtidos neste estudo permitem concluir que as dietas enterais apresentaram condições higiênico-sanitárias insatisfatórias em ambos os hospitais e que o manipulador é provavelmente, uma das fontes de maior significância para a contaminação da dieta enteral em ambiente hospitalar.