Understanding the role of Dimethylsulfoniopropionate as a Potential Chemotactic Cue for Coral-Associated Bacteria

Most reef-building corals are known to engage in symbiosis not only with unicellular dinoflagellates from the genus, Symbiodinium, but they also sustain highly complex symbiotic associations with other microscopic organisms such as bacteria, fungi, and viruses. The details of these non-pathogenic interactions remain largely unclear. The impetus of this study is to gain a better understanding of the symbiotic interaction between marine bacteria and a variety of coral species representative of differing morphologies. Studies have shown that certain bacterial orders associate specifically with certain coral species, thus making the symbiotic synergy a non-random consortium. Consequently both corals and bacteria may be capable of emitting chemical cues that enables both parties to find one another and thus creating the symbiosis. One potential chemical cue could be the compound DMSP (Dimethylsulfoniopropionate) and its sulphur derivatives. Reef-building corals are believed to be the major producers of the DMSP and its derivatives during times of stress. As a result corals could potentially attract their bacterial consortium depending on their DMSP production. Corals may be able to adapt to fluctuating environmental conditions by changing their bacterial communities to that which may aid in survival. The cause of this attraction may stem from the capability of a variety of marine bacteria to catabolize DMSP into different metabolically significant pathways, which may be necessary for the survival of these mutualistic interactions. To test the hypothesis that coral-produced DMSP play a role in attracting symbiotic bacteria, this study utilized the advent of high-through sequencing paired with bacterial isolation techniques to properly characterize the microbial community in the stony coral Porites astreoides. We conducted DMSP swarming and chemotaxis assays to determine the response of these coral-associated bacterial isolates towards the DMSP compound at differing concentrations. Preliminary data from this study suggests that six out of the ten bacterial isolates are capable of conducting unidirectional motility; these six isolates are also capable of conducting swarming motility in the direction of an increasing DMSP concentration gradient. This would indicate that there is a form of positive chemotaxis on behalf of the bacteria towards the DMSP compound. By obtaining a better understanding of the dynamics that drive the associations between bacterial communities and corals, we can further aid in the protection and conservation processes for corals. Also this study would further elucidate the significance of the DMSP compound in the survival of corals under times of stress.

Understanding the role of Dimethylsulfoniopropionate as a Potential Chemotactic Cue for Coral-Associated Bacteria

Most reef-building corals are known to engage in symbiosis not only with unicellular dinoflagellates from the genus, Symbiodinium, but they also sustain highly complex symbiotic associations with other microscopic organisms such as bacteria, fungi, and viruses. The details of these non-pathogenic interactions remain largely unclear. The impetus of this study is to gain a better understanding of the symbiotic interaction between marine bacteria and a variety of coral species representative of differing morphologies. Studies have shown that certain bacterial orders associate specifically with certain coral species, thus making the symbiotic synergy a non-random consortium. Consequently both corals and bacteria may be capable of emitting chemical cues that enables both parties to find one another and thus creating the symbiosis. One potential chemical cue could be the compound DMSP (Dimethylsulfoniopropionate) and its sulphur derivatives. Reef-building corals are believed to be the major producers of the DMSP and its derivatives during times of stress. As a result corals could potentially attract their bacterial consortium depending on their DMSP production. Corals may be able to adapt to fluctuating environmental conditions by changing their bacterial communities to that which may aid in survival. The cause of this attraction may stem from the capability of a variety of marine bacteria to catabolize DMSP into different metabolically significant pathways, which may be necessary for the survival of these mutualistic interactions. To test the hypothesis that coral-produced DMSP play a role in attracting symbiotic bacteria, this study utilized the advent of high-through sequencing paired with bacterial isolation techniques to properly characterize the microbial community in the stony coral Porites astreoides. We conducted DMSP swarming and chemotaxis assays to determine the response of these coral-associated bacterial isolates towards the DMSP compound at differing concentrations. Preliminary data from this study suggests that six out of the ten bacterial isolates are capable of conducting unidirectional motility; these six isolates are also capable of conducting swarming motility in the direction of an increasing DMSP concentration gradient. This would indicate that there is a form of positive chemotaxis on behalf of the bacteria towards the DMSP compound. By obtaining a better understanding of the dynamics that drive the associations between bacterial communities and corals, we can further aid in the protection and conservation processes for corals. Also this study would further elucidate the significance of the DMSP compound in the survival of corals under times of stress.

An investigation of the molecular interactions between statins and bacterial pathogens, and their combined impact on the human immune system

Statins are a class of drug that inhibits cholesterol biosynthesis, and are used to treat patients with high serum cholesterol levels. They exert this function by competitively binding to the enzyme 3-hydroxy-3-methylglutaryl-CoenzymeA reductase (HMGR), which catalyses the formation of mevalonate, a rate-limiting step in cholesterol biosynthesis. In addition, statins have what are called “pleiotropic effects”, which include the reduction of inflammation, immunomodulation, and antimicrobial effects. Statins can also improve survival of patients with sepsis and pneumonia. Cystic fibrosis (CF) is the most common recessive inherited disease in the Caucasian population, which is characterised by factors including, but not limited to, excessive lung inflammation and increased susceptibility to infection. Therefore, the overall objective of this study was to examine the effects of statins on CFassociated bacterial pathogens and the host response. In this work, the prevalence of HMGR was examined in respiratory pathogens, and several CF-associated pathogens were found to possess homologues of this enzyme. HMGR homology was analysed in Staphylococcus aureus, Burkholderia cenocepacia and Streptococcus pneumoniae, and the HMGR of B. cenocepacia was found to have significant conservation to that of Pseudomonas mevalonii, which is the most widely-characterised bacterial HMGR. However, in silico analysis revealed that, unlike S. aureus and S. pneumoniae, B. cenocepacia did not possess homologues of other mevalonate pathway proteins, and that the HMGR of B. cenocepacia appeared to be involved in an alternative metabolic pathway. The effect of simvastatin was subsequently tested on the growth and virulence of S. aureus, B. cenocepacia and S. pneumoniae. Simvastatin inhibited the growth of all 3 species in a dose-dependent manner. In addition, statin treatment also attenuated biofilm formation of all 3 species, and reduced in vitro motility of S. aureus. Interestingly, simvastatin also increased the potency of the aminoglycoside antibiotic gentamicin against B. cenocepacia. The impact of statins was subsequently tested on the predominant CF-associated pathogen Pseudomonas aeruginosa, which does not possess a HMGR homologue. Mevastatin, lovastatin and simvastatin did not influence the growth of this species. However, sub-inhibitory statin concentrations reduced the swarming motility and biofilm formation of P. aeruginosa. The influence of statins was also examined on Type 3 toxin secretion, quorum sensing and chemotaxis, and no statin effect was observed on any of these phenotypes. Statins did not appear to have a characteristic effect on the P. aeruginosa transcriptome. However, a mutant library screen revealed that the effect of statins on P. aeruginosa biofilm was mediated through the PvrR regulator and the Cup fimbrial biosynthesis genes. Furthermore, proteomic analysis demonstrated that 6 proteins were reproducibly induced by simvastatin in the P. aeruginosa swarming cells. The effect of statins on the regulation of the host-P. aeruginosa immune response was also investigated. Statin treatment increased expression of the pro-inflammatory cytokines IL-8 and CCL20 in lung epithelial cells, but did not attenuate P. aeruginosa-mediated inflammatory gene induction. In fact, simvastatin and P. aeruginosa caused a synergistic effect on CCL20 expression. The expression of the transcriptional regulators KLF2 and KLF6 was also increased by statins and P. aeruginosa, with the induction of KLF6 by simvastatin proving to be a novel effect. Interestingly, both statins and P. aeruginosa were capable of inducing alternative splicing of KLF6. P. aeruginosa was found to induce KLF6 alternative splicing by way of the type 3 secreted toxin ExoS. In addition, a mechanistic role was elucidated for KLF6 in the lung, as it was determined that statin-mediated induction of this protein was responsible for the induction of the host response genes CCL20 and iNOS. Moreover, statin treatment caused a slight increase in infection-related cytotoxicity, and increased bacterial adhesion to cells. Taken together, these data demonstrate that statins can reduce the virulence of CFassociated bacterial pathogens and alter host response effectors. Furthermore, novel statin effectors were identified in both bacterial and host cells.

Direct In Vivo Manipulation and Imaging of Calcium Transients in Neutrophils Identify a Critical Role for Leading-Edge Calcium Flux.

Calcium signaling has long been associated with key events of immunity, including chemotaxis, phagocytosis, and activation. However, imaging and manipulation of calcium flux in motile immune cells in live animals remain challenging. Using light-sheet microscopy for in vivo calcium imaging in zebrafish, we observe characteristic patterns of calcium flux triggered by distinct events, including phagocytosis of pathogenic bacteria and migration of neutrophils toward inflammatory stimuli. In contrast to findings from ex vivo studies, we observe enriched calcium influx at the leading edge of migrating neutrophils. To directly manipulate calcium dynamics in vivo, we have developed transgenic lines with cell-specific expression of the mammalian TRPV1 channel, enabling ligand-gated, reversible, and spatiotemporal control of calcium influx. We find that controlled calcium influx can function to help define the neutrophil's leading edge. Cell-specific TRPV1 expression may have broad utility for precise control of calcium dynamics in other immune cell types and organisms.

Influence of chylomicron remnants on human monocyte activation in vitro

Background and aims Atherosclerosis is known to be an inflammatory disease and there is increasing evidence that chylomicron remnants (CMR), the lipoproteins which carry dietary fats in the blood, cause macrophage foam cell formation and inflammation. In early atherosclerosis the frequency of activated monocytes in the peripheral circulation is increased, and clearance of CMR from blood may be delayed, however, whether CMR contribute directly to monocyte activation and subsequent egress into the arterial wall has not been established. Here, the contribution of CMR to activation of monocyte pro-inflammatory pathways was assessed using an in vitro model. Methods and results Primary human monocytes and CMR-like particles (CRLP) were used to measure several endpoints of monocyte activation. Treatment with CRLP caused rapid and prolonged generation of reactive oxygen species by monocytes. The pro-inflammatory chemokines MCP-1 and IL-8 were secreted in nanogram quantities by the cells in the absence of CRLP. IL-8 secretion was transiently increased after CRLP treatment, and CRLP maintained secretion in the presence of pharmacological inhibitors of IL-8 production. In contrast, exposure to CRLP significantly reduced MCP-1 secretion. Chemotaxis towards MCP-1 was increased in monocytes pre-exposed to CRLP and was reversed by addition of exogenous MCP-1. Conclusion Our findings indicate that CRLP activate human monocytes and augment their migration in vitro by reducing cellular MCP-1 expression. Our data support the current hypothesis that CMR contribute to the inflammatory milieu of the arterial wall in early atherosclerosis, and suggest that this may reflect direct interaction with circulating blood monocytes.

Cloning and Characterization of Two Potent Kunitz Type Protease Inhibitors from Echinococcus granulosus

The tapeworm Echinococcus granulosus is responsible for cystic echinococcosis (CE), a cosmopolitan disease which imposes a significant burden on the health and economy of affected communities. Little is known about the molecular mechanisms whereby E. granulosus is able to survive in the hostile mammalian host environment, avoiding attack by host enzymes and evading immune responses, but protease inhibitors released by the parasite are likely implicated. We identified two nucleotide sequences corresponding to secreted single domain Kunitz type protease inhibitors (EgKIs) in the E. granulosus genome, and their cDNAs were cloned, bacterially expressed and purified. EgKI-1 is highly expressed in the oncosphere (egg) stage and is a potent chymotrypsin and neutrophil elastase inhibitor that binds calcium and reduced neutrophil infiltration in a local inflammation model. EgKI-2 is highly expressed in adult worms and is a potent inhibitor of trypsin. As powerful inhibitors of mammalian intestinal proteases, the EgKIs may play a pivotal protective role in preventing proteolytic enzyme attack thereby ensuring survival of E. granulosus within its mammalian hosts. EgKI-1 may also be involved in the oncosphere in host immune evasion by inhibiting neutrophil elastase and cathepsin G once this stage is exposed to the mammalian blood system. In light of their key roles in protecting E. granulosus from host enzymatic attack, the EgKI proteins represent potential intervention targets to control CE. This is important as new public health measures against CE are required, given the inefficiencies of available drugs and the current difficulties in its treatment and control. In addition, being a small sized highly potent serine protease inhibitor, and an inhibitor of neutrophil chemotaxis, EgKI-1 may have clinical potential as a novel anti-inflammatory therapeutic.

Cellular and molecular phenotypes depending upon the RNA repair system RtcAB of Escherichia coli

RNA ligases function pervasively across the three kingdoms of life for RNA repair, splicing and can be stress induced. The RtcB protein (also HSPC117, C22orf28, FAAP and D10Wsu52e) is one such conserved ligase, involved in tRNA and mRNA splicing. However, its physiological role is poorly described, especially in bacteria. We now show in Escherichia coli bacteria that the RtcR activated rtcAB genes function for ribosome homeostasis involving rRNA stability. Expression of rtcAB is activated by agents and genetic lesions which impair the translation apparatus or may cause oxidative damage in the cell. Rtc helps the cell to survive challenges to the translation apparatus, including ribosome targeting antibiotics. Further, loss of Rtc causes profound changes in chemotaxis and motility. Together, our data suggest that the Rtc system is part of a previously unrecognised adaptive response linking ribosome homeostasis with basic cell physiology and behaviour.

The regulatory role of eNOS-derived nitric oxide on transcription in endothelial cells: Impact of S-nitrosylation on β-catenin signaling

Les cellules endothéliales forment une couche semi-perméable entre le sang et les organes. La prolifération, la migration et la polarisation des cellules endothéliales sont essentielles à la formation de nouveaux vaisseaux à partir de vaisseaux préexistants, soit l’angiogenèse. Le facteur de croissance de l’endothélium vasculaire (VEGF) peut activer la synthase endothéliale du monoxyde d’azote (eNOS) et induire la production de monoxyde d’azote (NO) nécessaire pour la régulation de la perméabilité vasculaire et l’angiogenèse. β- caténine est une composante essentielle du complexe des jonctions d’ancrage ainsi qu’un régulateur majeur de la voie de signalisation de Wnt/β-caténine dans laquelle elle se joint au facteur de transcription TCF/LEF et module l’expression de nombreux gènes, dont certains sont impliqués dans l’angiogenèse. La S-nitrosylation (SNO) est un mécanisme de régulation posttraductionnel des protéines par l’ajout d’un groupement nitroso au niveau de résidus cystéines. Le NO produit par eNOS peut induire la S-nitrosylation de la β−caténine au niveau des jonctions intercellulaires et moduler la perméabilité de l’endothélium. Il a d’ailleurs été montré que le NO peut contrôler l’expression génique par la transcription. Le but de cette thèse est d’établir le rôle du NO au sein de la transcription des cellules endothéliales, spécifiquement au niveau de l’activité de β-caténine. Le premier objectif était de déterminer si la SNO de la β-caténine affecte son activité transcriptionnelle. Nous avons montré que le NO inhibe l’activité transcriptionnelle de β- caténine ainsi que la prolifération des cellules endothéliales induites par l’activation de la voie Wnt/β-caténine. Il est intéressant de constater que le VEGF, qui induit la production de NO via eNOS, réprime l’expression de AXIN2 qui est un gène cible de Wnt s’exprimant suite à la i i stimulation par Wnt3a et ce, dépendamment de eNOS. Nous avons identifié que la cystéine 466 de la β-caténine est un résidu essentiel à la modulation répressive de son activité transcriptionnelle par le NO. Lorsqu’il est nitrosylé, ce résidu est responsable de la perturbation du complexe de transcription formé de β-caténine et TCF-4 ce qui inhibe la prolifération des cellules endothéliales induite par la stimulation par Wnt3a. Puisque le NO affecte la transcription, nous avons réalisé l’analyse du transcriptome afin d’obtenir une vue d’ensemble du rôle du NO dans l’activité transcriptionnelle des cellules endothéliales. L’analyse différentielle de l’expression des gènes de cellules endothéliales montre que la répression de eNOS par siRNA augmente l’expression de gènes impliqués au niveau de la polarisation tels que : PARD3A, PARD3B, PKCZ, CRB1 et TJ3. Cette analyse suggère que le NO peut réguler la polarisation des cellules et a permis d’identifier des gènes responsables de l’intégrité des cellules endothéliales et de la réponse immunitaire. De plus, l’analyse de voies de signalisation par KEGG montre que certains gènes modulés par l’ablation de eNOS sont enrichis dans de nombreuses voies de signalisation, notamment Ras et Notch qui sont importantes lors de la migration cellulaire et la différenciation des cellules de têtes et de tronc (tip/stalk). Le regroupement des gènes exprimés chez les cellules traitées au VEGF (déplétées de eNOS ou non) révèle que le NO peut affecter l’expression de gènes contribuant au processus angiogénique, dont l’attraction chimiotactique. Notre étude montre que le NO module la transcription des cellules endothéliales et régule l’expression des gènes impliqués dans l’angiogenèse et la fonction endothéliale.

Investigation of the roles of CheD in the Bacillus subtilis chemotactic signal transduction

Abstract The two-component based chemotaxis signal transduction system allows flagellated bacteria to sense their surrounding chemical environment and move towards more favorable conditions. The attractant signals can be sensed by transmembrane chemoreceptors, and then transmitted to the histidine kinase CheA. Once activated, CheA interacts with the response regulator CheY through phosphorelay, which causes a change in the rotation of the flagella. The direction of flagella rotation determines whether a cell swims straight or just tumbles. Cells also need adaptation to respond to a change in chemical concentrations, and return to their prestimulated level. Adaptation in the B. subtilis chemotaxis system is achieved by three coordinated systems: the methylation system, the CheC/CheD/CheY-p system and the CheV system. CheD, the previously identified receptor deamidase, was shown to be critical to the ability of B. subtilis to perform chemotaxis and is the main focus of this study. This study started from characterization of the enzymatic mechanism of CheD. Results showed that CheD deamidase uses a cysteine hydrolase mechanism. The catalytic triad consisting of Cys33-His50-Thr27, and Ser27 is essential for receptor recognition and binding. In addition, in this study CheC was found to inhibit CheD’s deamidase activity. Through mutant screening, Phe102 on CheD was found to be the essential site to interact with CheC. Furthermore, the CheD/CheC interaction is necessary for the robust chemotaxis in vivo as demonstrated by the cheD (F102E) mutant, which lacks the ability to swim on swarm plates. Despite its deamidase activity, we hypothesized that CheD’s main role is its involvement in the CheD-CheC-CheY-p negative feedback pathway during adaptation. In particular, CheD is likely to help stabilize the transient kinase-activating state through binding to receptors. When CheY-p level is increased, CheC-CheY-p complex may attract CheD away from receptors. In this study, CheC-CheD binding kinetics with CheY or CheYp presence was successfully obtained by a series of SPR experiments. The increased affinity of CheD for CheC in presence of CheYp but not CheY makes likely the hypothesis that CheC-CheD-CheY interact as part of a negative feedback pathway during adaptation. Last, the interaction between CheD and chemoreceptor McpC was studied in order to better understand the role of CheD in adaptation. Results showed that Q304 and Q305 on McpC are essential to recruit CheD. Additionally, the reduced levels of CheD in mcpC (Q304A) or (Q305A) mutants suggested that the dynamic interaction between CheD and receptors is vital to maintain the normal CheD level. These findings suggest more complicated roles of CheD than its previously identified function as a receptor deamidase, and will lead to a clearer picture of the coordination of the three adaptational systems in the B. subtilis chemotactic sensory transduction system.

Internal mammary artery smooth muscle cells resist migration and possess high antioxidant capacity

Objective- This study investigated whether differences exist in atherogen-induced migratory behaviors and basal antioxidant enzyme capacity of vascular smooth muscle cells (VSMC) from human coronary (CA) and internal mammary (IMA) arteries. Methods- Migration experiments were performed using the Dunn chemotaxis chamber. The prooxidant [NAD(P)H oxidase] and antioxidant [NOS, superoxide dismutase, catalase and glutathione peroxidase] enzyme activities were determined by specific assays. Results- Chemotaxis experiments revealed that while both sets of VSMC migrated towards platelet-derived growth factor-BB (1-50 ng/ml) and angiotensin II (1-50 nM), neither oxidized-LDL (ox-LDL, 25-100 �g/ml) nor native LDL (100 �g/ml) affected chemotaxis in IMA VSMC. However, high dose ox-LDL produced significant chemotaxis in CA VSMC that was inhibited by pravastatin (100 nM), mevastatin (10 nM), losartan (10 nM), enalapril (1 �M), and MnTBAP (a free radical scavenger, 50��M). Microinjection experiments with isoprenoids i.e. geranylgeranylpyrophosphate (GGPP) and farnesylpyrophosphate (FPP) showed distinct involvement of small GTPases in atherogen-induced VSMC migration. Significant increases in antioxidant enzyme activities and nitrite production along with marked decreases in NAD(P)H oxidase activity and O2 .- levels were determined in IMA versus CA VSMC. Conclusions- Enhanced intrinsic antioxidant capacity may confer on IMA VSMC resistance to migration against atherogenic agents. Drugs that regulate ox-LDL or angiotensin II levels also exert antimigratory effects.

Genome-scale analysis of the non-cultivable Treponema pallidum reveals extensive within-patient genetic variation

Insights into the genomic adaptive traits of Treponema pallidum, the causative bacterium of syphilis, have long been hampered due to the absence of in vitro culture models and the constraints associated with its propagation in rabbits. Here, we have bypassed the culture bottleneck by means of a targeted strategy never applied to uncultivable bacterial human pathogens to directly capture whole-genome T. pallidum data in the context of human infection. This strategy has unveiled a scenario of discreet T. pallidum interstrain single-nucleotide-polymorphism-based microevolution, contrasting with a rampant within-patient genetic heterogeneity mainly targeting multiple phase-variable loci and a major antigen-coding gene (tprK). TprK demonstrated remarkable variability and redundancy, intra- and interpatient, suggesting ongoing parallel adaptive diversification during human infection. Some bacterial functions (for example, flagella- and chemotaxis-associated) were systematically targeted by both inter- and intrastrain single nucleotide polymorphisms, as well as by ongoing within-patient phase variation events. Finally, patient-derived genomes possess mutations targeting a penicillin-binding protein coding gene (mrcA) that had never been reported, unveiling it as a candidate target to investigate the impact on the susceptibility to penicillin. Our findings decode the major genetic mechanisms by which T. pallidum promotes immune evasion and survival, and demonstrate the exceptional power of characterizing evolving pathogen subpopulations during human infection.

FAM49 - A novel regulator of the protrusive behaviour and motility of cells

Most eukaryotic cell motility relies on plasma membrane protrusions, which depend on the actin cytoskeleton and its tight regulation. The SCAR/WAVE complex, a pentameric assembly comprising SCAR/WAVE, Nap1, CYFIP/Pir121, Abi and HSPC300, is a key driver of actin-based protrusions such as pseudopods. SCAR/WAVE is thought to activate the Arp2/3 complex, a crucial actin nucleator, after being itself activated by upstream signals such as active Rac1. Despite recent progress on the study of the SCAR/WAVE complex, its regulation is still incompletely understood, with Nap1’s role being particularly enigmatic. Upon screening for potential Nap1 binding partners in the social amoeba Dictyostelium discoideum – a well established model organism in the study of the actin cytoskeleton and cell motility – we found FAM49, a ~36 kDa protein of unknown function which is highly conserved in Metazoa (animals) and evolutionarily closer species such as D. discoideum. Interestingly, D. discoideum’s FAM49 and its homologs contain a DUF1394 domain, which is also predicted in CYFIP/Pir121 proteins and most likely involved in their direct binding to active Rac1, which in turn contributes to SCAR/WAVE’s activation. FAM49’s unknown role, apparent high degree of conservation and potential connections to SCAR/WAVE and Rac1 persuaded us to start investigating its function and biological relevance in D. discoideum, leading to the work presented in this thesis. Several pieces of our data collectively support a function for FAM49 in modulating the protrusive behaviour, and ultimately motility, of D. discoideum cells, as well as a regulatory link between FAM49 and Rac1. FAM49’s involvement in protrusion regulation was first hinted at by our observation that GFP-tagged FAM49 is enriched in pseudopods. The possibility of a link with Rac1 was then strengthened by two additional observations: first, pseudopodial GFP-FAM49 is substantially co-enriched with active Rac, both showing fairly comparable spatio-temporal accumulation dynamics; second, when dominant-active (G12V) Rac1 is expressed in cells, it triggers the recruitment and persistent accumulation of GFP-FAM49 at the plasma membrane, where both become highly co-enriched. We subsequently determined that fam49 KO cells differ from wild-type cells in the way they protrude and move, as assessed in under-agarose chemotaxis assays. In particular, our data indicate that fam49 KO cells tend to display a lower degree of global protrusive activity, their protrusions extend more slowly and are less discrete, and the cells end up moving at lower speeds and with higher directional persistence. This phenotype was substantially rescued by FAM49 re-expression. While re-expressing FAM49 in fam49 KO cells we generated putative FAM49 overexpressor cells; compared to wild-type cells, they displayed atypically thin pseudopods and what seemed to be an excessively dynamic, and perhaps less coordinated, protrusive behaviour. Additional data in our study suggest that pseudopods made by fam49 KO cells are still driven by SCAR/WAVE, which is clearly not being replaced by WASP (as is now known to be the case in D. discoideum cells lacking a functional SCAR/WAVE complex). Nonetheless, the peculiar dynamics of those pseudopods imply that SCAR/WAVE’s activity is regulated differently when FAM49 is lost, though it remains to be determined how. This thesis is the first report of a dedicated study on FAM49 and lays the foundation for future research on it.

Resposta de Chromobacterium Violaceum cultivada em alta concentração de ferro

The Chromobacterium violaceum is a β-proteobacterium Gram-negative widely found in tropical and subtropical regions, whose genome was sequenced in 2003 showing great metabolic versatility and biotechnological and pharmaceutical potential. Given the large number of ORFs related to iron metabolism described in the genome of C. violaceum, the importance of this metal for various biological processes and due to lack of data about the consequences of excess of iron in free-living organisms, it is important to study the response mechanism of this bacterium in a culture filled with iron. Previous work showed that C. violaceum is resistant to high concentrations of this metal, but has not yet been described the mechanism which is used to this survival. Thus, to elucidate the response of C. violaceum cultured in high concentrations of iron and expecting to obtain candidate genes for use in bioremediation processes, this study used a shotgun proteomics approach and systems biology to assess the response of C. violaceum grown in the presence and absence of 9 mM of iron. The analysis identified 531 proteins, being 71 exclusively expressed by the bacteria grown in the presence of the metal and 100 just in the control condition. The increase in expression of proteins related to the TCA cycle possibly represents a metabolic reprogramming of the bacteria caused by high concentration of iron in the medium. Moreover, we observed an increase in the activity assay of superoxide dismutase and catalase as well as in Total Antioxidant Activity assay, suggesting that the metal is inducing oxidative stress in C. violaceum that increases the levels of violacein and antioxidant enzymes to better adapt to the emerging conditions. Are also part of the adaptive response changes in expression of proteins related to transport, including iron, as well as an increased expression of proteins related to chemotaxis response, which would lead the bacteria to change the direction of its movement away from the metal. Systems Biology results, also suggest a metabolic reprogramming with mechanisms coordinated by bottleneck proteins involved in transcription (GreA), energy metabolism (Rpe and TpiA) and methylation (AhcY)

High hydrostatic pressure adaptive strategies in an obligate piezophile Pyrococcus yayanosii

Pyrococcus yayanosii CH1, as the first and only obligate piezophilic hyperthermophilic microorganism discovered to date, extends the physical and chemical limits of life on Earth. It was isolated from the Ashadze hydrothermal vent at 4,100 m depth. Multi-omics analyses were performed to study the mechanisms used by the cell to cope with high hydrostatic pressure variations. In silico analyses showed that the P. yayanosii genome is highly adapted to its harsh environment, with a loss of aromatic amino acid biosynthesis pathways and the high constitutive expression of the energy metabolism compared with other non-obligate piezophilic Pyrococcus species. Differential proteomics and transcriptomics analyses identified key hydrostatic pressure-responsive genes involved in translation, chemotaxis, energy metabolism (hydrogenases and formate metabolism) and Clustered Regularly Interspaced Short Palindromic Repeats sequences associated with Cellular apoptosis susceptibility proteins.

The regulatory role of eNOS-derived nitric oxide on transcription in endothelial cells: Impact of S-nitrosylation on β-catenin signaling

Les cellules endothéliales forment une couche semi-perméable entre le sang et les organes. La prolifération, la migration et la polarisation des cellules endothéliales sont essentielles à la formation de nouveaux vaisseaux à partir de vaisseaux préexistants, soit l’angiogenèse. Le facteur de croissance de l’endothélium vasculaire (VEGF) peut activer la synthase endothéliale du monoxyde d’azote (eNOS) et induire la production de monoxyde d’azote (NO) nécessaire pour la régulation de la perméabilité vasculaire et l’angiogenèse. β- caténine est une composante essentielle du complexe des jonctions d’ancrage ainsi qu’un régulateur majeur de la voie de signalisation de Wnt/β-caténine dans laquelle elle se joint au facteur de transcription TCF/LEF et module l’expression de nombreux gènes, dont certains sont impliqués dans l’angiogenèse. La S-nitrosylation (SNO) est un mécanisme de régulation posttraductionnel des protéines par l’ajout d’un groupement nitroso au niveau de résidus cystéines. Le NO produit par eNOS peut induire la S-nitrosylation de la β−caténine au niveau des jonctions intercellulaires et moduler la perméabilité de l’endothélium. Il a d’ailleurs été montré que le NO peut contrôler l’expression génique par la transcription. Le but de cette thèse est d’établir le rôle du NO au sein de la transcription des cellules endothéliales, spécifiquement au niveau de l’activité de β-caténine. Le premier objectif était de déterminer si la SNO de la β-caténine affecte son activité transcriptionnelle. Nous avons montré que le NO inhibe l’activité transcriptionnelle de β- caténine ainsi que la prolifération des cellules endothéliales induites par l’activation de la voie Wnt/β-caténine. Il est intéressant de constater que le VEGF, qui induit la production de NO via eNOS, réprime l’expression de AXIN2 qui est un gène cible de Wnt s’exprimant suite à la i i stimulation par Wnt3a et ce, dépendamment de eNOS. Nous avons identifié que la cystéine 466 de la β-caténine est un résidu essentiel à la modulation répressive de son activité transcriptionnelle par le NO. Lorsqu’il est nitrosylé, ce résidu est responsable de la perturbation du complexe de transcription formé de β-caténine et TCF-4 ce qui inhibe la prolifération des cellules endothéliales induite par la stimulation par Wnt3a. Puisque le NO affecte la transcription, nous avons réalisé l’analyse du transcriptome afin d’obtenir une vue d’ensemble du rôle du NO dans l’activité transcriptionnelle des cellules endothéliales. L’analyse différentielle de l’expression des gènes de cellules endothéliales montre que la répression de eNOS par siRNA augmente l’expression de gènes impliqués au niveau de la polarisation tels que : PARD3A, PARD3B, PKCZ, CRB1 et TJ3. Cette analyse suggère que le NO peut réguler la polarisation des cellules et a permis d’identifier des gènes responsables de l’intégrité des cellules endothéliales et de la réponse immunitaire. De plus, l’analyse de voies de signalisation par KEGG montre que certains gènes modulés par l’ablation de eNOS sont enrichis dans de nombreuses voies de signalisation, notamment Ras et Notch qui sont importantes lors de la migration cellulaire et la différenciation des cellules de têtes et de tronc (tip/stalk). Le regroupement des gènes exprimés chez les cellules traitées au VEGF (déplétées de eNOS ou non) révèle que le NO peut affecter l’expression de gènes contribuant au processus angiogénique, dont l’attraction chimiotactique. Notre étude montre que le NO module la transcription des cellules endothéliales et régule l’expression des gènes impliqués dans l’angiogenèse et la fonction endothéliale.