A mesocosm experiment was carried out in a Norwegian fjord near Bergen in May 2006, with the main objective being the study of the effects of increasing concentrations of atmospheric CO2 (and associated effects such as increased acidification) on blooms of natural marine coastal plankton. Three mesocosms were bubbled with CO2(g) to achieve a high (~700ppm) CO2 concentration (pH ~7.8) to simulate predicted future conditions as a result of rising atmospheric CO2 concentrations. Another three mesocosms were treated as controls and bubbled with ambient air to represent a near pre-industrial scenario (atmospheric CO2 concentration ~300ppm, surface seawater pH ~8.15). Blooms in the mesocosms were stimulated by the addition of nutrients at a near-Redfield ratio ([N:P] ≈ [16:1]), and scientific measurements and analyses were carried out over the course of the blooms for approximately one month. Of particular interest in this study were the autotrophic plankton. The diversity and activities of these microorganisms under the two treatments was therefore investigated. By designing and using new degenerate primers specifically targeting ‘Green-type’ (Form IA and IB), ‘Red-type’ (Form IC and ID) and Form II RuBisCO, analysis of primary producers was carried out using PCR and either gDNA or cDNA (mRNA) templates from key time points spanning the complete duration of the blooms throughout the mesocosm experiment. Over 1250 novel RuBisCO large subunit sequences have been fully annotated and deposited in the NCBI GenBank® database. These sequences revealed distinct changes in the diversity of primary producers both over the courses of the blooms and between treatments. Particularly striking was the effect of acidification on the community structure of the eukaryotic picoplankton, Prasinophytes. A clade of prasinophytes closely related to Micromonas pusilla showed a distinct preference for the high CO2 conditions; a laboratory-based experiment confirmed the high tolerance of Micromonas pusilla to lower pH. Conversely, a clade related to Bathycoccus prasinos was almost entirely excluded from the high CO2 treatments. Clades of form II RuBisCO-containing dinoflagellates were also abundant throughout the experiment in both treatments. The high similarity of some of these clades to the toxin-producing species Heterocapsa triquetra and Gonyaulax polyedra, and apparent high tolerance of some clades to high CO2 conditions, is perhaps cause for concern in a high CO2 world and demands further research. In parallel with the RubisCO work, new primers were designed that target the gene encoding the Fe protein of nitrogenase (NifH). 82 Bergen genomic nifH sequences have been annotated and submitted to GenBank®. These sequences include those from organisms related to Alpha, Beta, and Gammaproteobacteria, and Cluster II and Cluster III sequences that align most closely with anaerobic Bacteria, Gram positive, and/or sulphur-reducing Bacteria. The biggest surprise, however, was the apparent abundance and significance of a Rhodobacter sphaeroides-like microorganism throughout the duration of the experiment in both treatments. Whilst this clade was unsurprisingly absent in the RuBisCO cDNA libraries, all but two of 128 nifH cDNA clones analysed were identical to the gene from Rhodobacter sphaeroides. This shows that this clade was potentially fixing N2 throughout the entire experiment, even in the presence of combined N added to both sets of mesocosms at the start of the experiment. A group of Rhodobacter sphaeroides-like microorganisms present at Bergen may therefore have been an unexpected source of new N during the experiment and contributed to the maintenance of the mesocosm communities as nutrients became depleted. One organism dominated the autotrophic communities after the blooms in both treatments. Synechococcus spp. Form IA rbcL clones most closely related to the coastal strain Synechococcus sp. strain CC9902 were recovered throughout the experiment but were particularly numerous toward the end of the experiment and dominated the “Green-type” libraries at this time. Initially, rbcL clones from these cyanobacteria were mostly derived from the ambient CO2 mesocosms but were equally distributed between treatments by the end of the experiment. This suggests that cyanobacteria related to strain CC9902 may be less tolerant of elevated CO2 (which was greatest at the beginning rather than the end of the experiment). However, despite the mesocosms being Pi-limited at the end of the experiment, several Synechococcus species (including those related to strain CC9902 and another coastal strain, CC9311) thrived. Following on from this observation, Pi uptake and assimilation mechanisms in a Synechococcus species were investigated in the laboratory. This led to the sequencing and characterisation of a pstS gene from the marine cyanobacterium Synechococcus sp. WH 8103. Unlike conventional pstS, it was discovered that the pstS II gene in this organism is constitutively expressed and unresponsive to or only weakly regulated by Pi supply. The use of PstS/pstS as a marker for P-limitation in natural samples, therefore, should be interpreted with caution.

L’intégration du génome du virus papilloma humain (VPH) a été reconnu jusqu’`a récemment comme étant un événnement fréquent mais pourtant tardif dans la progression de la maladie du col de l’utérus. La perspective temporelle vient, pourtant, d’être mise au défi par la détection de formes intégrées de VPH dans les tissus normaux et dans les lésions prénéoplasiques. Notre objectif était de déterminer la charge virale de VPH-16 et son état physique dans une série de 220 échantillons provenant de cols uterins normaux et avec des lésions de bas-grade. La technique quantitative de PCR en temps réel, méthode Taqman, nous a permis de quantifier le nombre de copies des gènes E6, E2, et de la B-globine, permettant ainsi l’évaluation de la charge virale et le ratio de E6/E2 pour chaque spécimen. Le ratio E6/E2 de 1.2 ou plus était suggestif d’intégration. Par la suite, le site d’intégration du VPH dans le génome humain a été déterminé par la téchnique de RS-PCR. La charge virale moyenne était de 57.5±324.6 copies d'ADN par cellule et le ratio E6/E2 a évalué neuf échantillons avec des formes d’HPV intégrées. Ces intégrants ont été amplifiés par RS-PCR, suivi de séquençage, et l’homologie des amplicons a été déterminée par le programme BLAST de NCBI afin d’identifier les jonctions virales-humaines. On a réussi `a identifier les jonctions humaines-virales pour le contrôle positif, c'est-à-dire les cellules SiHa, pourtant nous n’avons pas detecté d’intégration par la technique de RS-PCR dans les échantillons de cellules cervicales exfoliées provenant de tissus normaux et de lésions de bas-grade. Le VPH-16 est rarement intégré dans les spécimens de jeunes patientes. / Integration of human papillomavirus (HPV) has, until recently, been a frequent but late event in cervical carcinogenesis. The temporal view has, however, been challenged lately as integrated forms of HPV have been detected even in normal and preneoplastic lesions. Our objective was to describe HPV 16 load and physical state in a series of 220 normal and low grade cervical samples. We used quantitative real-time PCR, Taqman method, targeting E6, E2 and B-globin to calculate the HPV 16 load and the E6/E2 ratio in each sample. An E6/E2 ratio of 1.2 was used as a surrogate marker of integration. The site of integration was determined by restriction site PCR. Results show that the average viral load was 57.5±324.6 copies of DNA per cell, while E6/E2 ratio identified 9 samples with integrants. These integrants underwent amplification by restriction site PCR, followed by sequencing and nucleotide blast to identify the human-viral junctions. In conclusion, although it was possible to identify viral-host junctions with the integration positive control, that is, the SiHa cell line, the exfoliated cells of normal and low grade cervical lesions were negative for integration site by RS-PCR. HPV-16 is seldom integrated in specimens from young patients.

Aeromonas spp. são bastonetes Gram negativos amplamente distribuídos nos ambientes aquáticos, com relatos de isolamento em água de abastecimento público e alimentos. Este micro-organismo possui potencial de causar doenças intestinais e extraintestinais cuja patogenicidade está associada a sua virulência multifatorial. Diversos determinantes de virulência de Aeromonas já foram identificados, incluindo sistemas de secreção de proteínas. O sistema de secreção tipo VI (SST6) é o mais recente sistema de secreção de proteínas identificado em bactérias cuja presença em estirpes no gênero Aeromonas pode implicar atividades de citotoxicidade para o hospedeiro, pois esse sistema é capaz de injetar moléculas efetoras dentro da célula, interferindo diretamente nos processos celulares. A fim de determinar a presença e analisar a distribuição dos genes hcp e vgrG codificadores das proteínas efetoras do SST6 em Aeromonas spp. o presente estudo examinou 119 cepas isoladas de diversas origens pela técnica da PCR após o desenho de oligonucleotídeos iniciadores específicos. Objetivamos ainda analisar a variabilidade genética interespecífica dos genes hcp e vgrG a partir de dados de sequenciamento. Os resultados obtidos indicaram a distribuição dos genes vgrG e hcp em 46% das cepas de Aeromonas hydrophila e Aeromonas caviae de diferentes origens. Entre as cepas de A. hydrophila a maior frequência foi observada nas cepas isoladas de humanos, onde todas foram positivas para os iniciadores que amplificaram um produto de 541 pb do gene vgrG e 418 pb do gene hcp. Entre as cepas de A. caviae, a incidência de genes vgrG e hcp foi mais elevada nas cepas isoladas de alface (60%) e peixes (50%). As cepas analisadas de origem ambiental apresentaram índice total de 36% de positividade, apresentando frequência de 60% e 22% em A. hydrophila e A. caviae, respectivamente. Os dados obtidos da análise de cepas de origem alimentar mostraram a presença dos genes vgrG e hcp em 67% (A. hydrophila) e 60% (A. caviae) das cepas isoladas de folhas de alface. Nas cepas isoladas de queijo os genes foram encontrados em 67% e 12,5% das cepas de A. hydrophila e de A. caviae, respectivamente. O alinhamento múltiplo entre as sequências dos segmentos dos genes hcp e vgrG obtidas no sequenciamento indicou grau de identidade nucleotídica de 75 a 100% entre as sequências de hcp e 80 a 100% entre as sequências de vgrG. Em conclusão, nossos resultados indicaram que os iniciadores desenhados foram capazes de detectar suas sequências alvo em cepas de A. caviae e outras espécies de Aeromonas, sugerindo a existência de homologia entre os genes nas diferentes espécies, confirmada após sequenciamento de DNA. Os dados indicaram que esses genes estão distribuídos em várias espécies de Aeromonas e em cepas isoladas de diversas fontes. Ressaltamos a prevalência de cepas de A. hydrophila PCR-positivas em isolados clínicos, sugerindo a participação do SST6 no complexo universo da virulência multifatorial que permeia esse micro-organismo / Aeromonas species are Gram negative bacilli distributed widely in aquatic environments, with reports of isolation of this microorganism in water for public supply and food. Aeromonas have the potential to cause intestinal and extra intestinal infections whose pathogenicity is associated with multifactorial virulence. A number of virulence determinants have already been identified in Aeromonas, including protein secretion systems. The type VI secretion system (T6SS) is the most recent pathway to secrete proteins identified in bacteria. The presence of T6SS in Aeromonas strains may involve activities of cytotoxicity to the host, since this system is capable of injecting effectors molecules into the cell, interfering directly with a variety of cellular processes. The present study examined 119 strains of different origins by PCR, after the design of specific primers, to determine the distribution of vgrG and hcp genes encoding the effector proteins of T6SS in Aeromonas spp. We aimed to further analyze the interspecific sequence variation of hcp and vgrG genes based on sequencing data. The results show the presence of hcp and vgrG genes in 46% of A. hydrophila and A. caviae strains from different sources. All A. hydrophila strains isolated from humans were positive for the primers used to amplify a product of 541 bp and 418 bp of vgrG and hcp genes, respectively. Among A. caviae strains, the incidence of hcp and vgrG genes was high in the strains isolated from lettuce (60%) and fish (50%). The overall PCR-positive rate of strains from environmental source was 36%, with a frequency of 60% and 22% in A. hydrophila and A. caviae, respectively. The data obtained from analysis of food-borne strains showed the presence of hcp and vgrG genes in 67% (A. hydrophila) and 60% (A. caviae) of strains isolated from lettuce, while in the strains isolated from cheese the frequency was 67% (A. hydrophila) and 12.5% (A. caviae). The multiple alignment of hcp and vgrG sequences obtained revealed nucleotide identity rate between 75-100% among the hcp sequences and 80-100% in vgrG sequences. In conclusion, our results indicate that the primers designed were able to detect their target sequences in strains of A. caviae and other Aeromonas species, suggesting the existence of homology between genes in different species, as confirmed after DNA sequencing. The data indicate that these genes are distributed in various Aeromonas species from different sources. We emphasize the prevalence of PCR-positive A. hydrophila strains in clinical samples suggesting the involvement of T6SS in the complex universe of multifactorial virulence, which permeates this microorganism

L’intégration du génome du virus papilloma humain (VPH) a été reconnu jusqu’`a récemment comme étant un événnement fréquent mais pourtant tardif dans la progression de la maladie du col de l’utérus. La perspective temporelle vient, pourtant, d’être mise au défi par la détection de formes intégrées de VPH dans les tissus normaux et dans les lésions prénéoplasiques. Notre objectif était de déterminer la charge virale de VPH-16 et son état physique dans une série de 220 échantillons provenant de cols uterins normaux et avec des lésions de bas-grade. La technique quantitative de PCR en temps réel, méthode Taqman, nous a permis de quantifier le nombre de copies des gènes E6, E2, et de la B-globine, permettant ainsi l’évaluation de la charge virale et le ratio de E6/E2 pour chaque spécimen. Le ratio E6/E2 de 1.2 ou plus était suggestif d’intégration. Par la suite, le site d’intégration du VPH dans le génome humain a été déterminé par la téchnique de RS-PCR. La charge virale moyenne était de 57.5±324.6 copies d'ADN par cellule et le ratio E6/E2 a évalué neuf échantillons avec des formes d’HPV intégrées. Ces intégrants ont été amplifiés par RS-PCR, suivi de séquençage, et l’homologie des amplicons a été déterminée par le programme BLAST de NCBI afin d’identifier les jonctions virales-humaines. On a réussi `a identifier les jonctions humaines-virales pour le contrôle positif, c'est-à-dire les cellules SiHa, pourtant nous n’avons pas detecté d’intégration par la technique de RS-PCR dans les échantillons de cellules cervicales exfoliées provenant de tissus normaux et de lésions de bas-grade. Le VPH-16 est rarement intégré dans les spécimens de jeunes patientes. / Integration of human papillomavirus (HPV) has, until recently, been a frequent but late event in cervical carcinogenesis. The temporal view has, however, been challenged lately as integrated forms of HPV have been detected even in normal and preneoplastic lesions. Our objective was to describe HPV 16 load and physical state in a series of 220 normal and low grade cervical samples. We used quantitative real-time PCR, Taqman method, targeting E6, E2 and B-globin to calculate the HPV 16 load and the E6/E2 ratio in each sample. An E6/E2 ratio of 1.2 was used as a surrogate marker of integration. The site of integration was determined by restriction site PCR. Results show that the average viral load was 57.5±324.6 copies of DNA per cell, while E6/E2 ratio identified 9 samples with integrants. These integrants underwent amplification by restriction site PCR, followed by sequencing and nucleotide blast to identify the human-viral junctions. In conclusion, although it was possible to identify viral-host junctions with the integration positive control, that is, the SiHa cell line, the exfoliated cells of normal and low grade cervical lesions were negative for integration site by RS-PCR. HPV-16 is seldom integrated in specimens from young patients.

Aeromonas spp. são bastonetes Gram negativos amplamente distribuídos nos ambientes aquáticos, com relatos de isolamento em água de abastecimento público e alimentos. Este micro-organismo possui potencial de causar doenças intestinais e extraintestinais cuja patogenicidade está associada a sua virulência multifatorial. Diversos determinantes de virulência de Aeromonas já foram identificados, incluindo sistemas de secreção de proteínas. O sistema de secreção tipo VI (SST6) é o mais recente sistema de secreção de proteínas identificado em bactérias cuja presença em estirpes no gênero Aeromonas pode implicar atividades de citotoxicidade para o hospedeiro, pois esse sistema é capaz de injetar moléculas efetoras dentro da célula, interferindo diretamente nos processos celulares. A fim de determinar a presença e analisar a distribuição dos genes hcp e vgrG codificadores das proteínas efetoras do SST6 em Aeromonas spp. o presente estudo examinou 119 cepas isoladas de diversas origens pela técnica da PCR após o desenho de oligonucleotídeos iniciadores específicos. Objetivamos ainda analisar a variabilidade genética interespecífica dos genes hcp e vgrG a partir de dados de sequenciamento. Os resultados obtidos indicaram a distribuição dos genes vgrG e hcp em 46% das cepas de Aeromonas hydrophila e Aeromonas caviae de diferentes origens. Entre as cepas de A. hydrophila a maior frequência foi observada nas cepas isoladas de humanos, onde todas foram positivas para os iniciadores que amplificaram um produto de 541 pb do gene vgrG e 418 pb do gene hcp. Entre as cepas de A. caviae, a incidência de genes vgrG e hcp foi mais elevada nas cepas isoladas de alface (60%) e peixes (50%). As cepas analisadas de origem ambiental apresentaram índice total de 36% de positividade, apresentando frequência de 60% e 22% em A. hydrophila e A. caviae, respectivamente. Os dados obtidos da análise de cepas de origem alimentar mostraram a presença dos genes vgrG e hcp em 67% (A. hydrophila) e 60% (A. caviae) das cepas isoladas de folhas de alface. Nas cepas isoladas de queijo os genes foram encontrados em 67% e 12,5% das cepas de A. hydrophila e de A. caviae, respectivamente. O alinhamento múltiplo entre as sequências dos segmentos dos genes hcp e vgrG obtidas no sequenciamento indicou grau de identidade nucleotídica de 75 a 100% entre as sequências de hcp e 80 a 100% entre as sequências de vgrG. Em conclusão, nossos resultados indicaram que os iniciadores desenhados foram capazes de detectar suas sequências alvo em cepas de A. caviae e outras espécies de Aeromonas, sugerindo a existência de homologia entre os genes nas diferentes espécies, confirmada após sequenciamento de DNA. Os dados indicaram que esses genes estão distribuídos em várias espécies de Aeromonas e em cepas isoladas de diversas fontes. Ressaltamos a prevalência de cepas de A. hydrophila PCR-positivas em isolados clínicos, sugerindo a participação do SST6 no complexo universo da virulência multifatorial que permeia esse micro-organismo / Aeromonas species are Gram negative bacilli distributed widely in aquatic environments, with reports of isolation of this microorganism in water for public supply and food. Aeromonas have the potential to cause intestinal and extra intestinal infections whose pathogenicity is associated with multifactorial virulence. A number of virulence determinants have already been identified in Aeromonas, including protein secretion systems. The type VI secretion system (T6SS) is the most recent pathway to secrete proteins identified in bacteria. The presence of T6SS in Aeromonas strains may involve activities of cytotoxicity to the host, since this system is capable of injecting effectors molecules into the cell, interfering directly with a variety of cellular processes. The present study examined 119 strains of different origins by PCR, after the design of specific primers, to determine the distribution of vgrG and hcp genes encoding the effector proteins of T6SS in Aeromonas spp. We aimed to further analyze the interspecific sequence variation of hcp and vgrG genes based on sequencing data. The results show the presence of hcp and vgrG genes in 46% of A. hydrophila and A. caviae strains from different sources. All A. hydrophila strains isolated from humans were positive for the primers used to amplify a product of 541 bp and 418 bp of vgrG and hcp genes, respectively. Among A. caviae strains, the incidence of hcp and vgrG genes was high in the strains isolated from lettuce (60%) and fish (50%). The overall PCR-positive rate of strains from environmental source was 36%, with a frequency of 60% and 22% in A. hydrophila and A. caviae, respectively. The data obtained from analysis of food-borne strains showed the presence of hcp and vgrG genes in 67% (A. hydrophila) and 60% (A. caviae) of strains isolated from lettuce, while in the strains isolated from cheese the frequency was 67% (A. hydrophila) and 12.5% (A. caviae). The multiple alignment of hcp and vgrG sequences obtained revealed nucleotide identity rate between 75-100% among the hcp sequences and 80-100% in vgrG sequences. In conclusion, our results indicate that the primers designed were able to detect their target sequences in strains of A. caviae and other Aeromonas species, suggesting the existence of homology between genes in different species, as confirmed after DNA sequencing. The data indicate that these genes are distributed in various Aeromonas species from different sources. We emphasize the prevalence of PCR-positive A. hydrophila strains in clinical samples suggesting the involvement of T6SS in the complex universe of multifactorial virulence, which permeates this microorganism