Submitted by Rosangela Silva (rosangela.silva3@unioeste.br) on 2018-05-25T12:11:00Z No. of bitstreams: 2 Waleska Yana Lazaretti.pdf: 953419 bytes, checksum: 36f62c7ad6ece04a0ae6842c9d0df45c (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2018-05-25T12:11:00Z (GMT). No. of bitstreams: 2 Waleska Yana Lazaretti.pdf: 953419 bytes, checksum: 36f62c7ad6ece04a0ae6842c9d0df45c (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2018-03-08 / Acinetobacter baumannii (A. baumannii) is an important opportunistic, Gram-negative pathogen responsible for severe nosocomial infections such as pneumonia, septicemia, urinary tract infections and meningitis. Strains of A. baumannii have been identified in an endemic and epidemic manner in hospitals, being verified the occurrence of multiresistant strains in these environments, with important ability to adapt to selective changes and environmental pressures. Furthermore, multidrug resistance to antibiotics has been continuously studied because it is a global public health problem, resulting in failure of therapy, prolongation of hospitalization, increase of mortality and morbidity rates, and increase in the financial costs of treatment. This pathogen has varied strategies involved with antimicrobial resistance, but it is known that the bacteria are able to respond to unfavorable conditions in the medium through the rapid expression of heat shock proteins (HSP) and also appear to be involved with the stress response caused by the presence of antibiotics. Among the HSPs is ClpB, an ATP-dependent molecular chaperone belonging to the HSP100 family that is associated with several cellular activities, with the remarkable ability to rescue proteins damaged by stress. The objective of this work was to investigate the role of the clpB gene responsible for the coding of a heat shock protein through qPCR in response to stress generated by thermal shock and antibiotics in cells of a multidrug resistant strain of A. baumannii (RS4). Tests performed included analysis of the structure of the clpB gene with bioinformatics tools and analysis of the expression of the same gene by qRT-PCR in response to exposure to heat shock and subinhibitory concentrations of the following antibiotics: ampicillin (30 g mL-1 ), amoxicillin + sulbactam (12 g mL-1), cefepime (30 g mL-1), sulfamethoxazole + trimethoprim (120/8 g mL-1) and meropenem (18 g mL-1).The analysis of the qPCR results showed a transient increase in the induction of the clpB gene in the different treatments used in this study and repression of mRNA-clpB in the presence of cefepime. In addition, in the presence of ampicillin and amoxicillin associated with sulbactam the increase in mRNA-clpB synthesis was around 1.4 times higher after 20 min of incubation with the antibiotics than in the complete absence of the antibiotics. Surprisingly, in the presence of meropenen the induction of mRNA-clpB expression was more than 30-fold higher after 10 minutes of incubation with the antibiotic and more than 8-fold higher in the presence of sulfamethoxazole associated with trimetropin. These data suggest that A. baumannii through thermal stress and antibiotic exposure, adjusts transcription levels of gene clpB allowing the bacterium to survive unfavorable conditions of the medium. Consequently, it can be stated that the protein encoded by the clpB gene is an important virulence factor in response to antibiotics in this pathogen. / Acinetobacter baumannii (A. baumannii) é um importante patógeno oportunista, Gram-negativo e responsável por infecções nosocomiais severas como pneumonias, septicemias, infecções urinárias e meningites. Cepas de A. baumannii têm sido identificadas de maneira endêmica e epidêmica nos hospitais, sendo verificada a ocorrência de cepas multirresistentes nesses ambientes, com importante habilidade de adaptação a mudanças seletivas e pressões ambientais. Ainda, a multirresistência a antibióticos vem sendo estudada continuamente, por se tratar de um problema de saúde pública global, resultando em falha na terapia, no prolongamento da internação hospitalar, no aumento das taxas de mortalidade e morbidade e na elevação dos custos financeiros do tratamento. Esse patógeno possui estratégias variadas envolvidas com a resistência aos antimicrobianos, porém, sabe-se que as bactérias apresentam habilidade de responder a condições desfavoráveis do meio em que se encontram por meio da rápida expressão de proteínas de choque térmico (HSP) e parecem também estar envolvidas com a resposta a estresse causado pela presença de antibióticos. Dentre as HSPs, está a ClpB, chaperone molecular dependente de ATP, pertencente à família HSP100 que está associada a diversas atividades celulares, com a capacidade notável para resgatar proteínas danificadas pelo estresse. O objetivo deste trabalho foi investigar o papel do gene clpB em resposta a estresse gerado por choque térmico e antibióticos em células de uma cepa multirresistente de A. baumannii (RS4). Os testes realizados englobaram análise da estrutura do gene clpB com ferramentas de bioinformática e análise da expressão do mesmo gene por qRT-PCR em resposta à exposição a choque térmico e a concentrações subinibitórias dos seguintes antibióticos: ampicilina (30 g mL-1), amoxacilina+ sulbactam (12 g mL-1), cefepime (30 g mL-1), sulfametoxazol + trimetoprima (120/8 g/mL-1) e meropenem (18 g mL-1). Os resultados apontados por análise de bioinformática sugerem uma conservação da estrutura global de ClpB dentro do gênero Acinetobacter sp. A análise dos resultados de qPCR evidenciou aumento transitório na indução do gene clpB nos diferentes tratamentos utilizados neste estudo e repressão do mRNA-clpB na presença de cefepime. Em adição, tanto na presença de ampicilina como de amoxicilina associada à sulbactam o aumento na síntese de mRNA-clpB foi em torno de 1,4 vezes superior após 20 min de incubação com os antibióticos do que na completa ausência dos antibióticos. Surpreendentemente, na presença de meropenen, a indução da expressão do mRNA-clpB foi mais que 30 vezes superior após 10 minutos de incubação com o antibiótico e mais que 8 vezes superior na presença de sulfametoxaxol associado à trimetropina. Esses dados sugerem que A. baumannii, mediante estresse térmico e exposição a antibióticos, ajusta os níveis de transcrição do gene clpB, permitindo que a bactéria sobreviva a condições desfavoráveis do meio. Consequentemente, pode-se afirmar que a proteína codificada pelo gene clpB figura como importante fator de virulência em resposta a antibióticos neste patógeno.

The genus Bartonella contains Gram-negative arthropod-borne bacteria that are found in many small animal reservoirs and are capable of causing human disease. Bacteria utilize a general stress response system to combat stresses from their surrounding environments. In α-proteobacteria, the general stress response system uses an alternate σ factor as the main regulator and incorporates it with a two-component system into a unique system. Our study identifies the general stress response system in the α-proteobacterium, Bartonella henselae, where the gene synteny is conserved and both the PhyR and alternate σ factor have similar sequence and domain structures with other α-proteobacteria. Furthermore, we showed that the general stress response genes are up-regulated under conditions that mimic the cat flea vector. We also showed that both RpoE and PhyR positively regulate this system and that RpoE also affects transcription of genes encoding heme-binding proteins and the BadA adhesin. Finally, we also identified a histidine kinase, annotated as BH13820 that can potentially phosphorylate PhyR. In addition, analysis of the transcriptome from the Houston-1 strain of B. henselae by RNA-Seq reveals a family of small RNAs (termed Brt1-Brt9 for Bartonella Regulatory Transcripts 1-9) that may rapidly adapt gene expression patterns to the diverse hosts of this bacterium. This family of RNAs consists of nine novel, highly expressed intergenic transcripts, ranging from 193-205 nucleotides with a high degree of homology (70-100%) and stable predicted secondary structures that are unique to the genus Bartonella. Northern blot analysis indicates that transcription of these sRNAs was highest under conditions mimicking those of the cat flea vector (low temperature, high hemin). The predicted promoters for Brt1-Brt9 have been cloned upstream of a β-galactosidase reporter gene in pNS2 to identify conditions altering transcription. Immediately downstream of each of the nine putative sRNAs is a helix-turn-helix DNA binding protein (termed Trp1-9 for Transcriptional Regulatory Protein 1-9) that is poorly transcribed as determined by RNA-Seq. This gene organization is suggestive of a potential cis-acting RNA mechanism or riboswitch with the RNA secondary structure controlling transcription of the cognate downstream trp.

In fish, maternal stress prior to spawn has been reported to have effects on offspring phenotype. Cortisol, the main glucocorticoid (GC) stress hormone, has been proposed as a potential mediator of such effects because of its organizational role in early teleost development. The present thesis tested whether maternal social stress or treatment with cortisol (as a proxy for maternal stress) prior to spawn affects the cortisol response to stress and anxiety-related behaviours in offspring. In zebrafish (Danio rerio), offspring of dominant females exhibited greater boldness at 6 days post-fertilization (DPF). Interestingly, offspring of females that engaged in social interactions, regardless of the resulting social status of the two females, exhibited greater survival at 1 DPF, a greater fear-related decrease in activity in response to bright light at 6 DPF, and decreased baseline whole-body cortisol content at 0 and 30 DPF. A field experiment with wild largemouth bass (Micropterus salmoides) revealed that maternal cortisol treatment prior to spawn also affected offspring phenotype; offspring of cortisol-treated females had higher masses right after hatch, had greater fear responses, were less bold and less anxious, and exhibited an attenuated cortisol response to an acute stressor. Together, the results of the present thesis suggest that effects of maternal stress prior to spawn on offspring survival, growth, responses to stress, and anxiety-related behaviours are mediated, at least in part, by elevated maternal cortisol but not likely via increased deposition of maternal cortisol into eggs. The effects of maternal stress and cortisol treatment on offspring reported in the present thesis also suggest that maternal stress may prime offspring with adaptive traits to better survive in a stressful environment.

No description available.

Legionella pneumophila est un pathogène opportuniste avec un cycle de vie intracellulaire obligatoire, ils arrivent à se répliquer dans leurs cellules hôtes comme des protozoaires, en exploitant les protéines et les voies de signalisation de l’organisme infecté pour échapper à sa réponse immunitaire. L. pneumophila est décrite comme un organisme sans oscillations circadiennes, pourtant elle possède des gènes homologues aux gènes circadiennes kaiBC de cyanobactéries. En appliquant un système d’infections in vitro et in vivo ainsi qu’une approche de génomique comparative et fonctionnelle sur l’ organisme modèle Legionella pneumophila mon projet a pour objectif de caractériser le rôle des gènes kaiBC de L. pneumophila. Les protéines KaiABC de cyanobactéries encodent un oscillateur circadien permettant la coordination et l’optimisation temporelle de divers processus biologiques ainsi que l’adaptation aux fluctuations quotidiennes (comme la production d’oxygène via la photosynthèse pendant le jour et la fixation d’azote dans l’obscurité). Notre étude montre que kaiC, kaiB, avec le lpp1114 (codant pour une protéine à plusieurs domaines), l’ensemble constitue une unité transcriptionnelle sous la commande du facteur sigma RpoS. Les souches mutantes de l'opéron kai affichent une haute sensibilité sous conditions de stress par le paraquat ou le sel en comparaison avec la souche sauvage. En effect, nos données provenant d’une expérience utilisant des systèmes de double hybride suggèrent que les proteines KaiC et KaiB de L. pneumophila n’interagissent pas comme ceux de cyanobactéries. Cependant, une version étendue de L. pneumophila KaiB contenant des résidus de C-terminal de T. elongatus est capable d’interagir avec KaiC. Nous démontrons aussi que la structure cristalline de KaiB de L. pneumophila révèle un pliage pareil a ceux de thiorédoxine (protéine d'oxydoréduction) mais manque les résidus de l'extrémité C-terminale important pour l'interaction avec KaiC. En revanche, L. pneumophila KaiC conserve l'activité d'autophosphorylation, mais KaiB ne declénche pas la phosphorylation de KaiC comme chez les cyanobacteries. L'analyse phylogénétique des protéines de Kai indique qu'elles ont été transférés à L. pneumophila et ont évolué de Synechosystis KaiC2B2 et pas du copie circadienne KaiB1C1. Il semble que les protéines Kai de L. pneumophila améliorent son adaptation à des conditions stressantes et aux changements environnementaux. / Legionella pneumophila is an opportunistic pathogen with an intracellular life cycle that uses aquatic protozoa as replication niche and protection from harsh environments. Although L. pneumophila is not known to have a circadian clock, it encodes homologues of the KaiBC proteins of Cyanobacteria that regulate circadian gene expression. By using a wide range of in vitro, in vivo and in silico approaches I characterized the KaiB and KaiC proteins of L. pneumophila The proteins KaiABC of cyanobacteria coordinate a circadian oscillator that regulates many physiological functions in the cells according to the day and the night time induce by the rotation of the Earth (e.g. they do photosynthesis during the day and nitrogen fixation during the night). We show that L. pneumophila kaiB, kaiC and the downstream gene lpp1114, are transcribed as a unit under the control of the stress sigma factor RpoS. Mutant analyses revealed that the kai operon-encoded proteins increase fitness of L. pneumophila in competitive environments, and confer higher resistance to oxidative and sodium stress. Indeed, KaiC and KaiB of L. pneumophila do not interact as evidenced by yeast and bacterial two- hybrid analyses. Fusion of the C-terminal residues of cyanobacterial KaiB to Legionella KaiB restores their interaction. The crystal structure of L. pneumophila KaiB suggests that it is an oxidoreductase-like protein with a typical thioredoxin fold. In contrast, KaiC of L. pneumophila conserved autophosphorylation activity, but KaiB does not trigger the dephosphorylation of KaiC like in Cyanobacteria. The phylogenetic analysis indicates that L. pneumophila KaiBC resemble Synechosystis KaiC2B2 and not the circadian KaiB1C1 copy. Thus, the L. pneumophila Kai proteins do not encode a circadian clock, but enhance stress resistance and adaption to changes in the environments.

No description available.

Předložená disertační práce se zabývá studiem produkce a degradace polymerních materiálů s využitím mikroorganismů. Hlavní pozornost je upřena ke studiu produkce polyesterů bakteriálního původu - polyhydroxyalkanoátů. Tyto materiály jsou akumulovány celou řadou bakterií jako zásobní zdroj uhlíku, energie a redukční síly. Díky svým mechanickým vlastnostem, kterými silně připomínají tradiční syntetické polymery jako jsou polyetylén nebo polypropylén, a také díky své snadné odbouratelnosti v přírodním prostředí, jsou polyhydroxyalkanoáty považovány za ekologickou alternativu k tradičním plastům vyráběným z ropy. Polyhydroxyalkanoáty mají potenciál najít řadu aplikací v průmyslu, zemědělství ale také v medicíně. Významná část předložené práce je zaměřena na produkci polyhydroxyalkanoátů z odpadních substrátů pocházejících především z potravinářských výrob. Testována byla odpadní syrovátka nebo odpadní oleje z různých zdrojů. Právě využití levných odpadních substrátů je strategií, která by mohla přispět ke snížení ceny polyhydroxyalkanoátů a tím usnadnit jejich masové rozšíření. Podle výsledků dosažených v této práci jsou právě odpadní olejové substráty velice perspektivní cestou k ekonomicky rentabilní biotechnologické produkci polyhydroxyalkanoátů. Další část předložené práce se zabývá studiu spojení metabolické role polyhydroxyalkanoátů a stresové odpovědi bakterií. V této práci bylo zjištěno, že expozice bakteriální kultury řízené dávce etanolu nebo peroxidu vodíku významně navýší dosažené výtěžky a to o přibližně 30 %. Po aplikaci výše zmíněných stresových faktorů došlo k aktivaci metabolických drah vedoucí k odbourání stresového faktoru z média. Výsledkem bylo navýšení poměru NAD(P)H/NAD(P)+, což vedlo k částečné inhibici Krebsova cyklu a naopak aktivaci biosyntetické dráhy polyhydroxyalkanoátů. Mimoto došlo k významnému navýšení molekulové hmotnosti výsledných materiálů. Podle těchto výsledků se regulovaná aplikace vhodně zvolených stresových podmínek zdá být zajímavou strategií, která vede nejen k navýšení celkových výtěžků, ale také významnému zlepšení vlastností polymeru. Poslední část disertační práce se zabývala studiem procesu biodegradace polyuretanových materiálů. Polyuretanové eleastomery byly modifikovány rozličnými biopolymery za účelem navýšení jejich biodegradability. Tyto materiály byly posléze vystaveny působení směsné termofilní kultury jako modelového systému, který simuluje přirozené konsorcium bakterií. Přítomnost testovaných materiálů v kultivačním médiu vedla k neobvyklým růstovým charakteristikám bakteriální kultury. V průběhu prvních několika dní byl růst kultury silně inhibován, nicméně po překonání této neobvykle dlouhé lag-fáze došlo k intenzivnímu nárůstu kultury. Hlavní podíl na hmotnostním úbytku testovaných materiálů během experimentů měl samovolný rozpad materiálů, nicméně byl pozorován i vliv bakteriální kultury, kdy míra biotické degradace závisela na použitém modifikačním činidle. Nejvyšší míra biotické degradace byla pozorována u polyuretanového materiálu modifikovaného acetylovanou celulózou. Lag-fáze byla způsobena uvolněním nezreagovaného katalyzátoru (dibutylcínlaurát) a polyolu do kultivačního média. Bakteriální kultura se však po čase dokázala na přítomnost toxických látek v médiu adaptovat nebo je dokázala eliminovat.

The aim of the master thesis was to study the effect of the accumulation of polyhydroxyalkanoates (PHA) for bacterial resistance to selected stress factors. In the theoretical part the selected stress factors, polyhydroxyalkanoates and the involvement of polyhydroxyalkanoates into stress response of bacteria were reviewed. In the experimental part we used bacteria Cupriavidus necator H16 and its mutant strain Cupriavidus necator H16/PHB-4 unable of polyhydroxybutyrate (PHB) accumulation. The resistance of above-mentioned bacterial strains against thermal and osmotic stress was tested. According to the results of the experiment, when the bacteria were exposed to three different concentrations of NaCl (50, 100 and 200 g/l) PHB accumulating strain showed a higer resistance to hyperosmotic stress than the strain unable of PHB accumulation. There was demonstrated with Raman spectroscopy that in the hyperosmotic environment induced crystallization of the intracellular PHB granules. Transmission electron microscopy indicated that strain Cupriavidus necator H16/PHB-4 is subject to plasmolysis during hyperosmotic stress. As a consequence the hyperosmomotic stress occurs to the aggregation intracellular PHB granules in strain Cupriavidus necator H16 but there is no plasmolysis or is much less intensive.

La bactérie Deinococcus radiodurans se distingue par sa résistance exceptionnelle aux rayonnements γ, UV, à la dessiccation et au stress oxydant. La radiorésistance de D.radiodurans résulte de l’association de plusieurs mécanismes, dont des systèmes efficaces de réparation de l’ADN et de détoxification des ROS, la protection des protéines contre l’oxydation, une structure compacte du nucléoïde et des protéines spécifiques aux Deinococcaceae, qui sont fortement induites après l’exposition des cellules au rayonnement γ. Le gène ddrI (DNA damage response) est fortement induit après exposition des cellules au rayonnement γ et code un régulateur transcriptionnel appartenant à la sous-famille CRP (cAMP receptor protein). Comparée à la souche sauvage, la souche privée de DdrI présente des défauts de division cellulaire et/ou de ségrégation de l’ADN, et est sensible aux agents génotoxiques, au stress oxydant et au choc thermique. La prédiction in silico des cibles potentielles de DdrI suggère que cette protéine régule l’expression d’une centaine de gènes impliqués dans la réplication, la réparation de l’ADN, la transduction de signal, la réponse au stress oxydant et au choc thermique. La séquence consensus 5’TGTGA(N6)TCACA3’, extrapolée à partir des 115 séquences cibles potentielles de DdrI, est spécifiquement fixée par DdrI uniquement en présence d’AMPc. Après un choc thermique, DdrI induit directement ou indirectement l’expression de nombreux gènes codant des protéases, des protéines du métabolisme de l’ADN, des lipides, des carbohydrates ainsi qu’un inhibiteur de la traduction. PprA, une protéine spécifique aux Deinococcaceae, joue un rôle crucial dans la radiorésistance et est impliquée dans la ségrégation des chromosomes et/ou la division cellulaire après réparation de l’ADN. De manière intéressante, l’absence de RecN, une protéine de la famille SMC, supprime la sensibilité du mutant ΔpprA aux agents génotoxiques, aux inhibiteurs de l’ADN gyrase et les défauts de ségrégation observés dans le mutant ΔpprA après irradiation des cellules. Après exposition des cellules au rayonnement γ, l’absence de RecN réduit la fréquence de recombinaison entre ADN chromosomique et plasmidique, suggérant que RecN intervienne dans la réparation de l’ADN par recombinaison homologue. Nous proposons un modèle, dans lequel RecN, en favorisant la réparation de l’ADN par recombinaison homologue, nécessite la présence de PprA pour favoriser le recrutement des ADN topoisomérases et la résolution des contraintes topologiques engendrées par ce mécanisme de réparation d’ADN. / The Deinococcus radiodurans bacterium exhibits resistance to γ and UV radiation, desiccation and oxidative stress. The molecular mechanisms contributing to the radioresistance of D. radiodurans include very efficient DNA repair mechanisms and ROS detoxification systems, protein protection against oxidation, a compact nucleoid structure and a subset of Deinococcus specific genes which are strongly induced after γ radiation. The ddrI (DNA damage response) gene is highly up-regulated after exposure to γ radiation and encodes a transcription factor belonging to the CRP (cAMP receptor protein) family. Compared to wild type cells, cells devoid of DdrI display defects in cell division and/or DNA segregation and is sensitive to DNA damaging agents, oxidative stress and heat shock treatment. In silico predictions of putative DdrI targets suggest that hundreds of genes,belonging to various cellular processes (DNA replication and repair, oxidative stress and heat shock responses, regulation of transcription and signal transduction) may be regulated by DdrI. The pseudopalindromic 5’TGTGA(N6)TCACA3’ consensus sequence, extrapolated from 115 potential DdrI binding sites, is specifically bound by DdrI only in presence of cAMP. After heat shock treatment, DdrI is involved directly or indirectly, in the induction of heat shock response genes coding proteases, proteins involved in DNA, lipid, carbohydrate metabolism and a translation inhibitor. Among the Deinococcus specific proteins required for radioresistance, the PprA protein was shown to play a major role for accurate chromosome segregation and cell division after completion of DNA repair. Here, we analyzed the cellular role of the RecN protein belonging to the SMC family and, surprisingly, observed that the absence of the RecN protein suppressed the sensitivity of cells devoid of the PprA protein to γ- and UV-irradiation and to treatment with mitomycin C or DNA gyrase inhibitors. The absence of RecN also alleviated the DNA segregation defects displayed by the ΔpprA cells recovering from irradiation. After irradiation, the absence of RecN reduced recombination between chromosomal and plasmid DNA, indicating that the RecN protein is important for recombinational repair of DNA lesions. Here, we propose a model in which RecN, by favoring recombinational repair of DNA double strand breaks, requires the PprA protein to facilitate the recruitment of the DNA topoisomerases to resolve the topological constraints generated by DNA double strand break repair through homologous recombination.

We have previously characterized a Bacillus subtilis mutant defective in growth and osmoadaptation under limited K+ concentrations. In this mutant, the yxkO gene encoding a putative ribokinase is disrupted. This gene is supposed to belong to the sigma B operon and its expression is induced after osmotic, heat and ethanol shock. In comparison to the wild type, this mutation causes pleiotropic changes in host phenotype. In addition to its osmosensitivity, the mutant differs in cell shape, motility and ability to produce endospores. Our goal was to focus on manifestations of the mutation in the yxkO gene in other bacteria of the genus Bacillus. Using plasmid pMUTIN4 we have prepared mutants with disruptions of this gene derived from Bacillus amyloliquefaciens and Bacillus subtilis subsp. spizizenii strains differing in the yxkO surroundings and in the level of laboratory domestication. As in the previous study (with laboratory strain Bacillus subtilis 168) we demonstrate impaired ability of the mutant strain derived from Bacillus amyloliquefaciens to grow in potassium limitation and osmotic shock. We have studied this phenomenon at the level of the growth dynamics of the bacterial culture. We have also detected an increased sensitivity of the strain derived from Bacillus amyloliquefaciens to...