Bacillus subtilis, a model Gram-positive soil bacterium, employs two distinct mechanisms in its membrane adaptation to low temperature: 1) Long-term adaptation to suboptimal temperature is accomplished by increasing the ratio of anteiso- to iso-branched fatty acids in the membrane lipids. 2) After a sudden temperature decrease, the oxygen-dependent fatty acid desaturase (Des) is induced which desaturates fatty-acyl chains incorporated in membrane lipids. The transcription of the gene encoding desaturase, des, is activated by the decrease of the membrane order, via two- component system DesK-DesR. In this work, I studied the influence of cultivation conditions on the mechanisms of B. subtilis membrane adjustments for a low temperature employing fatty acid analysis, fluorescence spectroscopy, differential scanning calorimetry and methods of molecular biology. In the first part of this work, I examined the impact of the cultivation medium on the composition and biophysical features of the B. subtilis cytoplasmic membrane during growth under the optimal (40 řC) and suboptimal (20 řC) cultivation temperature. I compared the nutrient-rich complex medium containing glucose and the mineral medium supplemented with either glucose or glycerol. The results obtained showed the crucial importance of medium...

We investigated the effects of angiotensinogen (Ang), angiotensin I (Ang I), and angiotensin II (Ang II) on the fluidity of phosphatidylcholine vesicles. Changes in fluidity were assessed by changes in anisotopy values calculated from fluorescence polarization measurements. All three compounds produced an increase in membrane fluidity when localized inside the phosphatidylcholine vesicles. When placed outside the vesicles. Ang II increased bilayer rigidity (decreased fluidity), whereas Ang and Ang I produced no effect. These results suggest the possibility that these peptides may alter the fluidity of cell membranes by a direct action on the phospholipid bilayer, which may in turn interfere with receptor-mediated effects.

Plasma membranes have a structural property, commonly referred to as membrane fluidity, that is compositionally regulated. The two main features of plasma membrane lipid composition that determine membrane fluidity are the ratio of cholesterol to phospholipids and the ratio of saturated to unsaturated fatty acids that are incorporated into the phospholipids. These ratios are determined, at least in part, by regulation of membrane lipid biosynthesis-particularly that of cholesterol and oleate. It now appears that cholesterol and oleate biosynthesis are feedback regulated by a common transcriptional mechanism which is governed by the maturation of the SREBP transcription factors. In this article, we briefly review our current understanding of transcriptional regulation of plasma membrane lipid biosynthesis by sterols and oleate. We also discuss studies related to the mechanism by which the physical state of membrane lipids signals the transcriptional regulatory machinery to control the rates of synthesis of these structural components of the lipid bilayer.

In this thesis optical microscopy techniques are used to consider aspects of viral and bacterial infections. In part 1, the physical effects of cytomegalovirus on lipid droplet dynamics in live cells are studied; in part 2, the effects of an antimicrobial peptide on the fluidity of model bacterial membranes are studied. The optical microscopy techniques used to study the effects of murine-cytomegalovirus (mCMV) on lipid droplets in live NIH/3T3 fibroblast cells in real-time are coherent anti- Stokes Raman scattering (CARS), two-photon fluorescence (TPF) and differential interference contrast (DIC) microscopies. Using a multimodal CARS and TPF imaging system, the infection process was monitored by imaging the TPF signal caused by a green fluorescent protein (GFP)-expressing strain of mCMV, where the amount of TPF detected allowed distinct stages of infection to be identified. Meanwhile, changes to lipid droplet configuration were observed using CARS microscopy. Quantitative analysis of lipid droplet numbers and size distributions were obtained from live cells, which showed significant perturbations as the infection progressed. The CARS and TPF images were acquired simultaneously and the experimental design allowed incorporation of an environmental control chamber to maintain cell viability. Photodamage to the live cell population was also assessed, which indicated that alternative imaging methods must be adopted to study a single cell over longer periods of time. To this end, DIC microscopy was used to study the lipid droplet dynamics, allowing lipid droplet motion to be tracked during infection. In this way, the effects of viral infection on the mobility and arrangement of the lipid droplets were analysed and quantified. It was found that the diffusion coefficient of the lipid droplets undergoing diffusive motion increased, and the droplets undergoing directed motion tended to move at greater speeds as the infection progressed. In addition, the droplets were found to accumulate and cluster in infected cells. The second part of this thesis presents a study on the effects of an antimicrobial peptide on model bacterial membranes. Giant unilamellar vesicles (GUVs) were produced as a simple model of E. Coli membrane using a 3:1 mixture of DPPC and POPG lipids. Incorporating Laurdan fluorescent dye into the lipid membrane of the GUVs allowed the membrane fluidity to be probed and visualised using TPF microscopy, whereby the fluidity was quantified by determining the general polarization (GP) values. Studying GUVs comprising single lipid and mixed lipid compositions over a temperature range from 25 C to 55 C enabled the lipid phase bands to be identified on the basis of GP value as gel phase and liquid crystalline phase. As such, the changes in lipid phase as a result of interaction with AMP were quantified, and phase domains were identified. It was found that the amount of liquid crystalline phase domains increased significantly as a result of AMP interaction.

Lintégrité de la structure et de la dynamique de la membrane plasmatique est essentielle à la fonction de la cellule. Cette intégrité peut être évaluée par la mesure de la fluidité membranaire globale, reflet de lensemble des mouvements des éléments membranaires au sein de la bicouche phospholipidique. Or lintégrité de la membrane est menacée, entre autre, par les modifications de la structure lipidique résultant de lipoperoxidations. Ces peroxidations lipidiques résultent des attaques radicalaires par des espèces oxygénées activées (EOA) produites lors dagression oxydante sur les acides gras membranaires. Nous posons lhypothèse que les conditions doxygénations extrêmes, qui peuvent être rencontrées lors dune anesthésie ou lors dun stress oxydant induit par lexercice chez le cheval, peuvent affecter la fluidité membranaire des érythrocytes et que ces variations peuvent être modulées par la modification de la structure membranaire du globule rouge par un supplément antioxydant oral adéquat. Lobjectif de ce travail est donc dévaluer les effets de différentes conditions doxygénation et doxydation in vitro (par contact avec différents mélanges gazeux), puis in vivo sous anesthésie générale (en faisant varier la fraction inspirée en oxygène) et à lexercice, et enfin dévaluer les effets dune supplémentation enrichie en acides gras de type oméga-3 sur la fluidité membranaire du globule rouge. Les faibles pressions partielles en oxygène dans le sang artériel (PaO2), obtenues in vitro par contact du sang avec un gaz anoxique et in vivo sous anesthésie par inspiration dair ambiant (<45mmHg et <60mmHg respectivement), nont pas eu deffet sur la fluidité ni sur la structure de la membrane érythrocytaire. On peut supposer que le stimulus est insuffisant ou que la protection de la membrane résulte dune capacité antioxydante du plasma et de défenses cellulaires suffisantes. Les pressions partielles élevées en oxygène dans le sang, obtenues in vitro par contact du sang avec de loxygène pur (PaO2>500mmHg), ont induit un stress oxydant modéré qui na pas affecté la structure phospholipidique de la membrane malgré la peroxidation des acides gras de type oméga-6. La fluidité membranaire na pas été affectée par ces facteurs. In vivo, les pressions partielles élevées en oxygène observées dans le sang (>200mmHg) ont été insuffisantes pour induire des peroxidations significatives et des modifications de la fluidité membranaire. En revanche, les valeurs élevées de PaO2 ont augmenté la sensibilité du sang à lhémolyse dans un premier temps, puis sa résistance 24 heures après un retour à la normoxie. Dans ces conditions aucun effet na été noté sur la viscosité du sang ni la perfusion musculaire. Par ailleurs, lexercice intense semble diminuer la fluidité membranaire du globule rouge chez le cheval de sport. Cette diminution sobserve dès 15 minutes après larrêt de lexercice et persiste 24 heures après. Il existe également des corrélations entre certains de ces marqueurs indirects et la fluidité membranaire. La supplémentation na pas eu deffet significatif direct sur lévolution de la fluidité membranaire observée au repos. Mais elle a pourtant influencé la structure de la membrane. En effet, la complémentation a induit une augmentation du pourcentage dacides gras de type oméga-3 contenus dans la membrane érythrocytaire ainsi que du ratio oméga-3/oméga-6 pendant la période de repos. Cela résulte de lincorporation sélective dans la membrane de lacide eicosapentaénoïque (EPA) et de lacide docosahéxaénoïque (DHA) apportés par voie orale. Mais aucune corrélation na été observée dans notre étude entre la composition en acides gras de la membrane et le marqueur de la fluidité membranaire. La supplémentation na pas eu deffet significatif direct sur lévolution de la fluidité membranaire observée à lexercice, mais en a limité la diminution immédiate. Il résulte des études menées que : les conditions doxygénation les plus extrêmes qui peuvent être rencontrées en conditions atmosphériques ne semblent pas affecter la fluidité de la membrane. En revanche, un exercice intense, associé à une demande énergétique accrue, peut induire une diminution de la fluidité membranaire en corrélation avec les marqueurs du stress oxydant. Des modifications de la structure membranaire en acides gras polyinsaturés à longue chaîne de type oméga-3 naffectent pas la fluidité membranaire mais modulent les effets du stress oxydant lors de lexercice. La fluidité membranaire des érythrocytes pourrait être considérée comme un marqueur direct du stress oxydant dans certaines conditions. Mais ce marqueur semble moins sensible et global que dautres marqueurs du stress cellulaire tels que le test dhémolyse ou la mesure de la concentration plasmatique de peroxydes lipidiques spécifiques. The maintenance of plasmatic membrane integrity is mandatory for cell function. This integrity can be assessed by the measurement of global membrane fluidity which is proportional to the whole rotational and lateral diffusion rates of membrane components within the phospholipid bilayer. Membrane integrity could be threatened by changes in lipid structure as a result of lipid peroxidation by free radical species during oxidative stress. We hypothesize that extreme oxygenation status present during anesthesia or during exercise-induced oxidative stress in the horse can alter erythrocyte membrane fluidity (EMF), and that these changes in fluidity depend on variations in erythrocyte membrane structure under the action of an appropriate oral anti-oxidant supplementation. The aims of the study was: to assess the effect(s) of various oxygenation and oxidative conditions firstly created in vitro (by contact between erythrocyte and different gaz mixtures), and secondly in vivo during general anesthesia (with varying inspired oxygen fractions) as well as during exercise. To assess the effects of an omega-3 fatty acid-enriched supplementation on EMF. Low partial oxygen pressures, both obtained in vitro and in vivo under anesthesia (respectively <45 and <60 mmHg) did not have any effect on EMF or membrane structure. Erythrocyte membrane may have been protected by an increase in plasmatic anti-oxidative capacity and cellular defenses. High partial oxygen pressures (>500 mm Hg) obtained in vitro induced a moderate oxidative stress which did not alter the phospholipidic structure of the membrane despite peroxidation of omega 6 fatty acids. Partial oxygen pressures obtained in vivo (>200 mm Hg) were unable to induce significant peroxidation and alteration in membrane fluidity. However, high PaO2 values initially increased sensitivity of blood to hemolysis, followed by a tendency towards resistance to hemolysis after 24hours. Intense exercise decreases EMF in the sports horse. This was observed as soon as 15 minutes after exercise and persisted during the recovery period 24 hours later. Correlations were found between oxidative stress indirect markers and membrane fluidity. Supplementation did not affect membrane fluidity but influenced membrane structure by increasing the pourcentage of omega-3 fatty acids and the omega3/omega6 ratio at rest. These changes resulted from selective incorporation into the membrane of orally provided EPA and DHA . However, we could not evidence a correlation between membrane composition and the marker of membrane fluidity (correlation-relaxation time Tc). During exercise, supplementation had no direct effect on variations of membrane fluidity but tapered its immediate decrease. In conclusion, our studies show that the most extreme conditions encountered under atmospheric conditions do not appear to affect EMF. However intense exercise combined with increased energetic requirements induces a decrease in EMF which correlates with variations in markers of oxidative stress. Modifications of membrane composition in long-chain omega-3 polyinsaturated fatty acids do not affect EMF but modulate oxidative stress during exercise. EMF could be a direct marker of oxidative stress under certain conditions but appears less sensitive and comprehensive than other markers of celllular stress such as the hemolysis test or the concentration in specific lipidic peroxidation products.

Examples of phenotypic plasticity—the ability of organisms of identical genotypes to produce different phenotypes in response to the environment—are abundant, but often lack data on the causative physiology and biochemistry. Phenotypes associated with increased protection against or reduced damage from harmful environments may, in fact, be downstream effects of hidden adaptive responses that remain elusive to experimental measurement or be obscured by homeostatic or over-compensatory effects. The freshwater zooplankton crustacean Daphnia drastically increases its heat tolerance as the result of acclimation to high temperatures, an effect often assumed to be based on plastic responses allowing better protection against oxidative stress. Using several geographically distant Daphnia magna genotypes, we demonstrate that the more heat tolerant individuals have a higher total antioxidant capacity (TAC) both in the comparison of heat-acclimated vs. non heat-acclimated females and in the comparison of females to age- and body size-matched males, which show lower heat tolerance than females. However, experimental manipulations of hypothesized antioxidant pathways by either glutathione addition or glutathione synthesis inhibition had no effect on heat tolerance. Lipid peroxidation (LPO), contrary to expectations, did not appear to be a predictive measure of susceptibility to thermal damage: LPO was higher, not lower, in more heat tolerant heat-acclimated individuals after exposure to a lethally high temperature. We hypothesize that LPO may be maintained in Daphnia at a constant level in the absence of acute exposure to elevated temperature and increase as a by-product of a possible protective antioxidant mechanism during such exposure. This conclusion is corroborated by the observed short-term and long-term changes in phospholipid composition that included an increase in fatty acid saturation at 28 °C and up-regulation of certain long-chain polyunsaturated fatty acids. Phospholipid composition was more strongly affected by recently experienced temperature (4-day transfer) than by long-term (2 generations) temperature acclimation. This is consistent with partial loss of thermal tolerance after a short-term switch to a reciprocal temperature. As predicted under the homeoviscous adaptation hypothesis, the more heat tolerant Daphnia showed lower membrane fluidity than their less heat tolerant counterparts, in comparison both between acclimation temperatures and among different genotypes. We conclude that thermal tolerance in Daphnia is influenced by total antioxidant capacity and membrane fluidity at high temperatures, with both effects possibly reflecting changes in phospholipid composition.

To reduce reliance on fossil fuels, sustainable biofuels are being pursued, especially advanced biofuels like 1-butanol that have higher energy content and greater compatibility with existing infrastructure than ethanol. A persistent challenge is the yield-limiting toxicity of biofuels and process solvents, such as tetrahydrofuran, to the microbes that ferment biomass into biofuel. The cell membrane is a focal point of toxicity, and understanding how it interacts with fuels and solvents is key to improving yield. Phospholipid bilayers are the core of biomembranes, and model biomembranes of defined composition provide the ideal platform for biophysical studies. To this end, glycerophospholipids characteristic of Bacillus subtilis, a model producer organism, were synthesized. Two fatty acids (iso- and anteisopentadecanoic acids) characteristic of Bacilli were synthesized and incorporated into representative phosphatidic acid, phosphatidylethanolamine and phosphatidylglycerol lipids. The validated synthetic approach opens the door to future studies on the interaction of biofuels and solvents with biomembranes.

Pseudomonas aeruginosa é uma proteobactéria do grupo gama muito versátil, capaz de colonizar ambientes variados e infectar hospedeiros filogeneticamente distintos, incluindo humanos imunocomprometidos. Os fatores sigma de função extracitoplasmática (ECF) são membros de sistemas de sinalização de superfície celular (CSS), abundantes em P. aeruginosa. Vinte genes codificando fatores sigma ECF estão presentes nos genomas sequenciados de P. aeruginosa, a maioria fazendo parte de sistemas TonB relacionados à captação de ferro. Neste trabalho, seis fatores sigma pobremente caracterizados foram superexpressos na linhagem PA14 a partir de um promotor induzível por arabinose para investigar seu papel na expressão dos sistemas de dois componentes PvrSR e RcsCB, que atuam na regulação da fímbria CupD, além de sua influência no crescimento de culturas de P. aeruginosa. Não foi observado efeito positivo de nenhum dos fatores sigma testados na expressão dos sistemas de dois componentes e a superexpressão de cinco deles tampouco levou a qualquer alteração no crescimento, porém a produção de piocianina foi alterada na superexpressão de PA14_55550 e a superexpressão de PA14_26600 e PA14_46810 levou a um discreto aumento no início da formação de biofilme em PA14. Por outro lado, culturas superexpressando &#963;x (ALB04) apresentaram um perfil alterado de lipopolissacarídeo e uma curva de crescimento bifásica, alcançando precocemente uma fase estacionária seguida de uma recuperação do crescimento até uma segunda fase estacionária. Durante a primeira fase estacionária, a maior parte das células aumenta de tamanho e morre, mas as células remanescentes retornam à morfologia selvagem e seguem para a segunda fase de crescimento exponencial. Isso não acontece devido a mutações compensatórias, uma vez que células coletadas de pontos tardios da curva e diluídas em meio novo repetem este comportamento. Apesar de trabalhos com a linhagem PAO1 associarem &#963;x à transcrição de oprF, que codifica a principal porina não específica de Pseudomonas, nas condições dos nossos ensaios em PA14 a expressão dessa porina não foi induzida pela superexpressão de &#963;x. Assim, os efeitos observados nessa superexpressão também não podem ser atribuídos a OprF. A transcrição de oprF em PA14 mostrou-se majoritariamente dependente da região promotora a que se atribui a ligação de &#963;70, ao contrário dos relatos na literatura da dependência da região de ligação a &#963;x. Análises proteômicas foram realizadas para investigar os elementos envolvidos nesses efeitos de superexpressão de &#963;x, o que revelou a indução de diversas enzimas envolvidas na via de biossíntese de ácidos graxos. As células superexpressando &#963;x apresentam uma maior proporção de ácidos hexadecanoico (C16) e hexadecenoico (C16:1) e dados de anisotropia mostram uma maior fluidez da(s) membrana(s). Este trabalho é o primeiro relato de um fator sigma ECF envolvido em biossíntese de lipídeos em P. aeruginosa. / Pseudomonas aeruginosa is a very versatile gammaproteobacteria, able to colonize different environments and to infect phylogenetically distinct hosts, including immunocompromised humans. The extracytoplasmic function sigma factors (ECFs) are members of cell signaling systems (CSS), abundant in P. aeruginosa. Twenty genes coding for ECF sigma factors are present in the sequenced genomes of P. aeruginosa, most of them being part of TonB systems related to iron uptake. In this work, six poorly characterized sigma factors were overexpressed in strain PA14 from an arabinose inducible promoter to investigate their role in the expression of the two-component systems PvrSR and RcsCB, which regulates CupD fimbria, and their influence in P. aeruginosa cultures growth. None of the tested sigma factors led to two-component systems upregulation and overexpression of five of them caused no change in the growth profile, but pyocyanin production was altered in PA14_55550 overexpression and PA14_26600 and PA14_46810 overexpression led to a slight increase in biofilm initiation in PA14. By the other side, cultures overexpressing &#963;x (ALB04) presented an altered lipopolysaccharide profile and a biphasic growth curve, reaching an early stationary phase followed by a growth resuming untill a second stationary phase. During the early stationary phase, most cells swells and dies, but the remaining cells return to wild type morphology and proceed to the second exponential phase of growth. This is not due to compensatory mutations, since cells collected from late points of the curve and diluted in fresh medium repeat this behavior. Although studies with strain PAO1 associate &#963;x with transcription of oprF, encoding the major nonspecific porin of Pseudomonas, under our experiments conditions with PA14, this porin expression is not induced by &#963;x overexpression. Thus, the effects observed in this overexpression cannot be attributed to OprF. Transcription of oprF in PA14 proved to be mainly controlled by the &#963;70-dependent promoter region instead of the &#963;x-dependent promoter region reported in the literature. Proteomic analyses were performed to investigate the elements involved in these effects of &#963;x overexpression, which revealed the induction of several enzymes involved in fatty acids biosynthesis. Cells overexpressing &#963;x exhibit a greater proportion of hexadecanoic (C16) and hexadecenoic (C16: 1) acids and anisotropy data show higher fluidity of the membrane (s). This work is the first report of an ECF sigma factor involved in lipid biosynthesis in P. aeruginosa.

Several lines of evidence link Alzheimer's disease (AD) to atherosclerosis (CVD), including that elevated low density lipoprotein (LDL)-cholesterol is a common risk factor. Development of genomic instability could also link the two diseases. Previous fluorescence in situ hybridization (FISH) analyses revealed a clonal expansion of aneuploid smooth muscle cells underlying atherosclerotic plaques. Likewise, cellular and mouse models of AD revealed tau-dependent mitotic defects and subsequent aneuploidy partly resulting from amyloid-beta (A&beta) interference with microtubule (MT) stability, and specific MT motors function. Moreover, AD patients develop aneuploid/hyperploid cells in brain and peripheral tissues, implicating similar mechanism that may lead to apoptosis and neurodegeneration. This dissertation tested the hypothesis that elevated lipoproteins and cholesterol may contribute to genomic instability in AD and CVD and showed that: (1) treatment with oxidized LDL (OX-LDL), LDL and water soluble cholesterol, but not high density lipoprotein (HDL), induced chromosome mis-segregation, including trisomy and tetrasomy 12, 21, and 7 in human epithelial cells (hTERT-HME1), primary aortic smooth muscle cells, fibroblasts, mouse splenocytes and neural precursors; (2) LDL-induced aneuploidy may depend on a functional LDL receptor (LDLR), but not amyloid precursor protein (APP) gene; (3) fibroblasts and brain cells of patient with the mutation in the Niemann-Pick C1 gene (NPC1) characterized by impaired intracellular cholesterol trafficking and changed intracellular cholesterol distribution harbored trisomy 21 cells; (4) young wild-type mice fed high and low cholesterol diets developed aneuploidy in spleen but not in brain cells within 12 weeks; (5) like with the studies on A&beta-induced aneuploidy, calcium chelation reduced OX-LDL and LDL-mediated chromosomal instability; and (6) altering plasma membrane fluidity with ethanol attenuated OX-LDL and LDL-induced aneuploidy. These results suggest a novel biological mechanism by which disrupted cholesterol homeostasis may promote both atherosclerosis and AD by inducing chromosome mis-segregation and development of aneuploid cells. Understanding the cause and consequence of chromosomal instability as a common pathological trait in AD and CVD may be beneficial to designing therapies relevant for both diseases.

Pseudomonas aeruginosa é uma proteobactéria do grupo gama muito versátil, capaz de colonizar ambientes variados e infectar hospedeiros filogeneticamente distintos, incluindo humanos imunocomprometidos. Os fatores sigma de função extracitoplasmática (ECF) são membros de sistemas de sinalização de superfície celular (CSS), abundantes em P. aeruginosa. Vinte genes codificando fatores sigma ECF estão presentes nos genomas sequenciados de P. aeruginosa, a maioria fazendo parte de sistemas TonB relacionados à captação de ferro. Neste trabalho, seis fatores sigma pobremente caracterizados foram superexpressos na linhagem PA14 a partir de um promotor induzível por arabinose para investigar seu papel na expressão dos sistemas de dois componentes PvrSR e RcsCB, que atuam na regulação da fímbria CupD, além de sua influência no crescimento de culturas de P. aeruginosa. Não foi observado efeito positivo de nenhum dos fatores sigma testados na expressão dos sistemas de dois componentes e a superexpressão de cinco deles tampouco levou a qualquer alteração no crescimento, porém a produção de piocianina foi alterada na superexpressão de PA14_55550 e a superexpressão de PA14_26600 e PA14_46810 levou a um discreto aumento no início da formação de biofilme em PA14. Por outro lado, culturas superexpressando &#963;x (ALB04) apresentaram um perfil alterado de lipopolissacarídeo e uma curva de crescimento bifásica, alcançando precocemente uma fase estacionária seguida de uma recuperação do crescimento até uma segunda fase estacionária. Durante a primeira fase estacionária, a maior parte das células aumenta de tamanho e morre, mas as células remanescentes retornam à morfologia selvagem e seguem para a segunda fase de crescimento exponencial. Isso não acontece devido a mutações compensatórias, uma vez que células coletadas de pontos tardios da curva e diluídas em meio novo repetem este comportamento. Apesar de trabalhos com a linhagem PAO1 associarem &#963;x à transcrição de oprF, que codifica a principal porina não específica de Pseudomonas, nas condições dos nossos ensaios em PA14 a expressão dessa porina não foi induzida pela superexpressão de &#963;x. Assim, os efeitos observados nessa superexpressão também não podem ser atribuídos a OprF. A transcrição de oprF em PA14 mostrou-se majoritariamente dependente da região promotora a que se atribui a ligação de &#963;70, ao contrário dos relatos na literatura da dependência da região de ligação a &#963;x. Análises proteômicas foram realizadas para investigar os elementos envolvidos nesses efeitos de superexpressão de &#963;x, o que revelou a indução de diversas enzimas envolvidas na via de biossíntese de ácidos graxos. As células superexpressando &#963;x apresentam uma maior proporção de ácidos hexadecanoico (C16) e hexadecenoico (C16:1) e dados de anisotropia mostram uma maior fluidez da(s) membrana(s). Este trabalho é o primeiro relato de um fator sigma ECF envolvido em biossíntese de lipídeos em P. aeruginosa. / Pseudomonas aeruginosa is a very versatile gammaproteobacteria, able to colonize different environments and to infect phylogenetically distinct hosts, including immunocompromised humans. The extracytoplasmic function sigma factors (ECFs) are members of cell signaling systems (CSS), abundant in P. aeruginosa. Twenty genes coding for ECF sigma factors are present in the sequenced genomes of P. aeruginosa, most of them being part of TonB systems related to iron uptake. In this work, six poorly characterized sigma factors were overexpressed in strain PA14 from an arabinose inducible promoter to investigate their role in the expression of the two-component systems PvrSR and RcsCB, which regulates CupD fimbria, and their influence in P. aeruginosa cultures growth. None of the tested sigma factors led to two-component systems upregulation and overexpression of five of them caused no change in the growth profile, but pyocyanin production was altered in PA14_55550 overexpression and PA14_26600 and PA14_46810 overexpression led to a slight increase in biofilm initiation in PA14. By the other side, cultures overexpressing &#963;x (ALB04) presented an altered lipopolysaccharide profile and a biphasic growth curve, reaching an early stationary phase followed by a growth resuming untill a second stationary phase. During the early stationary phase, most cells swells and dies, but the remaining cells return to wild type morphology and proceed to the second exponential phase of growth. This is not due to compensatory mutations, since cells collected from late points of the curve and diluted in fresh medium repeat this behavior. Although studies with strain PAO1 associate &#963;x with transcription of oprF, encoding the major nonspecific porin of Pseudomonas, under our experiments conditions with PA14, this porin expression is not induced by &#963;x overexpression. Thus, the effects observed in this overexpression cannot be attributed to OprF. Transcription of oprF in PA14 proved to be mainly controlled by the &#963;70-dependent promoter region instead of the &#963;x-dependent promoter region reported in the literature. Proteomic analyses were performed to investigate the elements involved in these effects of &#963;x overexpression, which revealed the induction of several enzymes involved in fatty acids biosynthesis. Cells overexpressing &#963;x exhibit a greater proportion of hexadecanoic (C16) and hexadecenoic (C16: 1) acids and anisotropy data show higher fluidity of the membrane (s). This work is the first report of an ECF sigma factor involved in lipid biosynthesis in P. aeruginosa.