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“Role of Autophagy in the regulation of Angiogenesis in Stroke”

“Role of Autophagy in the regulation of Angiogenesis in Stroke”

As shown in Figure 8, during the first step of Initiation, one of the drugs used for the activation of autophagy is the Metformin that is a well known pharmacological compound largely used to treat type 2 diabetes mellitus (DM2) and this drug is of particular interest for its ability to activate the AMPK cascade (Zhou, Myers et al. 2001, Viollet, Guigas et al. 2012, Wrobel, Marek et al. 2017). In fact, the autophagy activation by AMPK can be proceeded through two mechanisms: inhibition of mTOR or phosphorylation of ULK-1 (Steinberg and Kemp 2009, Villanueva-Paz, Cotan et al. 2016). In the phase following initiation, that is named nucleation, several researchers have shown that it can be inhibited by drugs as LY290042 or 3MA through an inhibition mechanism of the class III PI3K complex that is required for the formation of PAS (Caro, Plomp et al. 1988, Blommaart, Krause et al. 1997, Stroikin, Dalen et al. 2004, Wu, Tan et al. 2010, Zou, Zhang et al. 2014). More, in the step before the degradation, the lysosome is fused with the autophagosome exposing the organelles and compounds to the degradation caused by the action of the lysosomal hydrolases. In this phase, the Chloroquine acts with a mechanism of prevention of the endosomal acidification. It accumulates in the lysosomes causing an increase in lysosomal pH, which inhibits lysosome function and may block fusion of the autophagosome with the lysosome (Beil, Staar et al. 1992, Beil, Sewing et al. 1999). Or the Bafilomycin (Hanada, Moriyama et al. 1990, Oda, Nishimura et al. 1991) and Pantoprazole (Moreira Dias 2009, Vegger, Bruel et al. 2017) that are two inhibitors of the Vacuolar H + ATPase, which controls the pH in the lysosome and causing an elevated lysosomal
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Unravelling the role of NAADP/TPC2 Ca2+ signalling during angiogenesis and tumor progression

Unravelling the role of NAADP/TPC2 Ca2+ signalling during angiogenesis and tumor progression

In HUVECs, we have previously demonstrated that VEGF- dependent Ca 2+ mobilization evoked by NAADP is impaired pretreating cells with Ned-19 (a noncompetitive antagonist of NAADP) [1]. In order to evaluate the possible inhibitory effect of Nar on the regulation of VEGF-dependent Ca 2+ signalling in human cells, calcium imaging experiments were performed pretreating HUVECs for 30 min with different concentrations of Nar and stimulating them with 100 ng/ml VEGF. Figure 3a,b shows that Ca 2+ mobilization was significantly reduced in a dose- dependent manner without affecting VEGFR2 phosphorylation at Tyr1175 demonstrating that the inhibition of the responses occurs downstream of the receptor , (Figure 4). Furthermore, opting for 1000 µM Nar, for its highest effect, we have analyzed the specificity of this response for NAADP/TPC2- dependent Ca 2+ signalling using a positive and a negative control. Bar charts in Figure 3c,d show that Nar fails to block Ca 2+ response to ATP, known to be IP 3 -dependent, but blocks histamine-evoked Ca 2+
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The novel anti-IL17A monoclonal antibody Secukinumab in the treatment of psoriasis: biological effects on angiogenesis and extracellular vesicles

The novel anti-IL17A monoclonal antibody Secukinumab in the treatment of psoriasis: biological effects on angiogenesis and extracellular vesicles

Dysregulated angiogenesis is one of the key features of psoriasis. Many different growth factors and pro-inflammatory cytokines involved in the modulation of the angiogenic process are up-regulated in psoriasis development, such as fibroblast growth factor (FGF), angiopoietins, hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), epidermal growth factor (EGF), transforming growth factor-β (TGF-β), tumor necrosis factor- α (TNF-α), platelet-activating factor, ephrins, soluble adhesion molecules and angiogenin. Among these, the vascular endothelial growth factor (VEGFA) plays a major role in regulating endothelial cell functions, including proliferation, migration and activation (Lamalice L et al., 2007). IL-17A is known to regulate angiogenesis inducing the expression of VEGFA in keratinocytes. The VEGFA is upregulated in the psoriatic epidermis (Marina ME et al., 2015; Gerkowicz A et al., 2018). Thus, we evaluated by RT real-time PCR the expression levels of a panel of angiogenic factors, such as EGF, FGF, PDGF, TGF-β, TNF-α, including VEGFA, after rIL-17A treatment. In addition, the effect of the treatment with Secukinumab on the transcriptional levels of angiogenesis-related genes was also evaluated. To this purpose, HaCaT cells were treated for 24 hours with rIL-17A in presence or absence of Secukinumab; at the end of each treatment, cell culture derived supernatants were also collected, as described in Materials and methods, and tested by ELISA for the secreted angiogenic protein. We detected the up-regulation of EGF, FGF, PDGF, TGF-β and TNF-α mRNA levels after rIL-17A treatment, as expected. The pre-treatment with Secukinumab significantly reduced the induction of these mRNAs compared to rIL-17A-treated cells as well as Secukinumab alone. The treatment with rIL-17A increased the expression of VEGFA at both mRNA and secreted protein levels. Conversely, the pre-treatment with Secukinumab decreased the expression of VEGFA to control levels (Figure 9 A-B).
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Role of transglutaminase 2 in brain ageing

Role of transglutaminase 2 in brain ageing

As above mentioned, TG2 is a multifunctional protein involved in different cellular processes, particularly those involved in elderly age. The observed increase of mRNA levels during normal aging and TG2 role in apoptosis, autophagy, ROS imbalance e inflammation create a direct link with the ageing process. How tight is this link and how TG2 modulation may modify/improve the physiopathological aging, is yet to be understood. It is certain though that there are grounds for TG2 to become a new therapeutic target in many age-related diseases: cardiovascular disease for instance, seen the role in cardiovascular biology, including contributing to the development of hypertension, influencing the progression of atherosclerosis, regulating vascular permeability and angiogenesis and contributing to myocardial signaling, contractile activity and ischemia/reperfusion injury (Sane et al., 2007); cancer pathology, TG2 has indeed emerged as a putative gene involved in tumor cell drug resistance and evasion of apoptosis (Budillon et al., 2013); hepatic disease, since TG2 plays a protective role in the liver injury by favoring tissue stability and repair (Nardacci et al., 2003).
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Behavioral, physiological and neural evidence of the role of political ideology in social cognition

Behavioral, physiological and neural evidence of the role of political ideology in social cognition

statistical computing (R Development Core Team, 2013). Trials in which participants did not recognize the politician were excluded from the analysis (Total valid trials=62%; range: min=13%, max=100% of valid trials per subject). We then performed a multilevel mixed linear regression analysis (LMM or “mixed-effects models”; (Garson 2013; Pinheiro and Bates 2000) through the package lme4 Version 1.1–5 (Bates, D., Maechler, M., Bolker, B., & Walker 2014). Unlike traditional statistical methods, LMM are suitable for (a) analyzing hierarchical data structures (i.e., in which not all levels of a categorical factor co-occur at all levels of another categorical factor); (b) analyzing the whole data set (not just the mean observations for each subject and condition) to better evaluate the data variations that variance-style analyses (ANOVA) often leave out; (c) accounting for the non-independence of observations with correlated error; (d) separately treating the effects caused by the experimental manipulation (fixed effects) and those that were not (random effects) (Pinheiro and Bates 2000). We used Valence as the dependent measure of our model. The fixed effects were the Political Categorization of the stimulus, the Group and their respective interactions. Political categorization of the stimulus was recoded as follows: if the categorization of the stimulus made by the participant matched his/her Group (i.e., left vs right-wing), that stimulus was considered as ingroup (e.g., a stimulus categorized as left-wing by a left-wing participant was considered as ingroup). If the categorization did not match, that stimulus was considered as outgroup (e.g., a stimulus categorized as right-wing by a left-wing participant was considered as outgroup). We considered the random intercept over participants and the random slope of Political categorization over participants as random factors. Statistical significance of fixed effects was determined using type III Wald F tests with Kenward-Roger degrees of freedom (Kenward and Roger 1997) and the Anova function from R’s car package. Post hoc pairwise comparisons (FDR corrected) were performed using least squares contrasts (lsc), as employed in R’s lsmeans package. The analysis revealed a significant Group x Political Categorization interaction F (1, 70.025) = 48.31, p< .001. Post hoc analysis showed the slope of left-wing participants to be significantly different from zero (b= 3.11, SE= 0.26, df= 68.75, t.ratio= -11.94, p< .001), while that of right-wing
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Studying the role of chromatin remodelers during cell division

Studying the role of chromatin remodelers during cell division

Chromatin has hierarchical levels ranging from the repetition of the unit of basic, the nucleosome, at higher order levels (Ou et al., 2017). This organization, finely regulated, represents a dynamic balance between packaging of the genome and its accessibility. The linear length of DNA chromosome in a human cell presents a significant topological challenge since about 2 meters of DNA must be packed in the core characterized by a diameter of only 6 μm (Andrews and Luger, 2011; Khorasanizadeh, 2004). Nucleosomes are positioned along DNA in a “beads on a string” configuration to create a 10-nm fiber.
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<<An>> investigation on the usability of the Kinect by Microsoft in upper limb rehabilitation after stroke

<<An>> investigation on the usability of the Kinect by Microsoft in upper limb rehabilitation after stroke

algorithms, but a long term abilities evaluation is necessary to dynamically adapt. The manual setting by the therapist, adopted in this work, avoids preliminary training of performance detection algorithms, and allows having a system less standardized and more patient-oriented than automatic systems, that can be used with patients affected by different types of pathologies. The proposed software interface reduces the limitations of other systems, allowing the therapist to create new games without complex coding. Traditional rehabilitation practice is then maintained and the therapist has the key-role to define the rehabilitation plan for each patient, according to the specific needs. However, there seems to be still some issues in the easiness of designing patient-oriented video-games by the therapist, as highlighted in [109], where the proposed framework resulted to be difficult to use by one of the therapists. This study thus proposed a system which was developed in collaborations between engineers and therapists to let the therapist design a patient-oriented rehabilitation task in an easy, user- friendly way, without the need of any specific informatics skills. The system was thus made flexible at source and this allowed a more intuitive usage.
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Regulation of cell physiology through the modulation of estrogen receptors activities by natural and synthetic compounds

Regulation of cell physiology through the modulation of estrogen receptors activities by natural and synthetic compounds

cellular content control seems to rely on 26S proteasome mediated degradation, as both E2 as well as EDs induce the ERα mRNA levels down-regulation (fig 2C), while only the endogenous hormone and the EDs reduce ERα protein level (Fig. 2A and B) and the incubation with the proteasome inhibitor, MG-132, reverts the observed effect (Fig. 7C). As E2 and EDs produced the same regulation of ERα mRNA levels, we suspected that rapid E2-induced ERα signaling, and therefore ERα membrane localization, could also participate in ERα protein regulation. ERα is palmitoylated on the C447 by the action of two PAT and that the PAT-dependent enzymatic palmitoylation is required for ERα to associate with caveolin-1 and to mediate E2 extra-nuclear signaling [33, 38]. Our research group has also indicated that E2 binding determines ERα depalmitoylation and dissociation from caveolin-1, a series of mechanistic events that facilitate receptor movements within membrane subdomains [33]. As a consequence, E2 activation of the extra-nuclear signaling kinase cascades (e.g,. ERK/MAPK and PI3K/AKT pathways) occurs and regulates several different physiological processes (i.e., proliferation, apoptosis, and differentiation) [85]. Therefore, we used a genetic (i.e., mutation of the ERα palmitoylation site C447 to A) and a pharmacological (i.e., inhibition of PAT activity) approach to evaluate the impact of E2:ERα extra-nuclear signaling activation on E2-induced receptor degradation.
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Economic crisis, role of public authorities and the commons : Rome and the governance of urban resources

Economic crisis, role of public authorities and the commons : Rome and the governance of urban resources

To be more specific, we might recall with Sturn (2006) that Musgrave‘s perspective on collective goods originated also from Hermann‘s subjectvisim. According to Sturn, ―Musgrave gives impressive overviews of important writers of German Staatswirthschaft such as Hermann and the writers of the aforementioned ‗triad‘ as well as Sax, von Wieser, Wicksell, and Lindahl, who applied marginal utility analysis to the theory of public expenditure‖(Sturn 2006: 41). Hermann is the reference for a ―non-atomistic subjectivism‖ that provide a basis for Musgrave conception of State. Hermann emphasized the role of the subject of action and their development, and his subjectivism was not utilitarian, focusing on how in the process of socio-economic development individuals define and increase cognitive skills, motivations and needs.. According to this author, ―self-preservation was the basic motivational assumption and source of needs‖ (Sturn 2006:52). The family is the basic—social condition of self-preservation, since it primarily addresses specific needs, motivations and activities defining the spectrum of collective needs and joint consumption 20 . The process of civilization enlarges the objective of self-preservation and defines new collective needs beyond the level of the individual or the family. These collective (joint) needs (at least in Sturn‘s perspective) define collective goods. The necessity of this goods at a wider level motivate the intervention of the State and the public sector. Indeed, similarly to Musgrave, collective needs define collective goods that need the existence of State/government. Hermann discussed the conditions under which impure public goods are better supplied on a voluntary basis by some intermediate institutions sustained by the public spirit or the government 20 and Sturn reports in a passage by Hermann an envision of market and State that strongly reminds Musgrave‘s conception and also the literature by Adam Smith, Robbins, and Ropke. ‗Presupposing all the social means of securing and enhancing industry and trade, and assuming moreover sufficiently educated citizens, one may well admit that, as a rule, the individual‘s own advantage is the best guide to the kind and method of his industry, whereas constraints are rather harmful than useful‘ (Hermann (1832: 13))
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Regulation of vitamin B6 metabolism in Escherichia coli

Regulation of vitamin B6 metabolism in Escherichia coli

So far, no specific transcriptional regulators of PLP biosynthesis and salvage pathways have been found in E. coli. In Salmonella typhimurium, the expression of the pdxK gene is repressed from the MocR-like regulator PtsJ, whose gene is divergently transcribed. PLP acts as effector molecule of this regulator, because its binding to PtsJ induces a protein conformational change that increases affinity for DNA and reinforces repression (Tramonti et al. 2017). As stated above, in several bacteria, which synthesize PLP through the so-called DXP-independent pathway, the transcription of the pdxST operon encoding PLP synthase is regulated by another MocR-TF (PdxR). A similar transcriptional regulation of PLP biosynthesis has never been identified in E. coli, where the function of the two MocR-TFs present in the genome (Table 3.1) has never been identified. We also analysed the promoter region of all the genes and operons involved in PLP biosynthesis and salvage pathways, to check for the presence of common motifs that may correspond to transcriptional regulation binding sites. A 13-bp conserved sequence was identified in all the 18 promoter regions analysed, with pdxJ, pdxK, tyrS and
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The right to food: alternative methods of implementation: The Role of Courts and Indicators

The right to food: alternative methods of implementation: The Role of Courts and Indicators

sity to protect individuals from starvation. 121 In this way, it conferred to what was treated as a mere aspirational objective the validity of a legal entitlement. On the one hand, the case is a paradigmatic example of a right to food approach brought around through bottom-up agitation. The large-scale Right to Food Campaign led by PUCL did a terrific job in keeping the victims posted on the devel- opments and maintaining a steady flow of communication between the civil society and the PUCL Commission created ad hoc by the Court and task with analysing policy data from both the federal and the State level. The Court paid great heed to its reports and often cited them verbatim. Indeed, the construction of such triangular relationship created a united front which could highlight any shortcoming in policy implementation and communicate it promptly to the Commission, which at- tempted to find a settlement with the government before turning the issue to the Court. On the other hand, the case is illustrative of the role of courts and justiciability in contrasting the effects of the international neoliberal paradigm on the right to food. Indeed, the Court’s provisions were in many cases contrary to the liberalising aims of the World Bank- and IMF-sponsored New Economic Poli- cy (NEP), as well as of the WTO regulations to which India is subject, deemed greatly harmful for internal food security in a country where 60% of the population rely on agriculture for their liveli- hoods, the majority being rural smallholder farmers. The Court stepped in and corrected many measures that had been taken under the structural adjustments; in particular, ordering the govern- ment to increase spending in order to ensure rural employment was essentially an agricultural sub- sidy, in contrast with the AoA. By acting as a “floor to Indian Capitalism”, PUCL humanised it by refusing to allow people to starve as a short-term cost of adjustment of liberalisation for mere utili- tarian calculation (the prize of a higher aggregate wealth later on). It did so by using human rights as rigid and unsurmountable barriers that must not be crossed by the action of the State, no matter the objective or competing interest. The usage of a right to food approach to economic policy is recognisable in the bottom-up movement caused and funnelled by PUCL, making the grievances of the victims heard, instead of merely receiving it in a top-down fashion, once again proving that it is through the existence of courts able to apply human rights that democracy is preserved and devel- opment can take a human visage. 122
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Effects of adiponectin on the progression of breast cancer: role of Estrogen Receptor alpha

Effects of adiponectin on the progression of breast cancer: role of Estrogen Receptor alpha

Breast cancer is stimulated by estrogens that activating the classical ERs modulate cell proliferation, adhesion, migration, and invasion in estrogen-sensitive tumors (2, 3). In addition, estrogens promote the formation of new blood vessels within the tumor mass (35), hence suggesting that these steroids elicit a stimulatory role not only in cancer cells but also in compo- nents of the surrounding stroma in accordance with the results obtained in this study. As the complex process of angiogenesis is required for tumor progression, it represents a central biological target in cancer (36). VEGF is one of the most potent proangiogenic factor playing a paramount role in the forma- tion of blood vessels in the development of different types of tumors, including breast cancer (23). Accordingly, VEGF is highly expressed in breast cancer specimens compared with normal breast tissue (37) and its suppression leads to the
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TBX1 Transcription Factor: mechanisms of gene regulation

TBX1 Transcription Factor: mechanisms of gene regulation

The gene studied in the thesis work was identified in an effort to isolate the gene/s involved in DiGeorge syndrome (DGS) or 22q11.2 deletion syndrome (22q11.2DS) (Linsday EA et al., 2001; Merscher S et al., 2001). Congenital heart disease (CHD) affects 8/1000 live births. A common genetic cause of CHD is the 22q11.2 deletion, also known as DiGeorge syndrome (DGS) (McDonald-McGinn DM et al., 2015). It has been estimated that a substantial portion of patients with some specific heart defects have 22q11.2 deletions: 52% of those with interrupted aortic arch type B, 34% with truncus arteriosus, 16% with tetralogy of Fallot and 5-10% with Ventricular Septal Defects (VSD). Besides CHD, patients have a number of other phenotypic features, for example cleft palate, development disabilities and schizophrenia. At the genetic level, the deletion could result from aberrant homologous recombination between low copy repeat (LCR) sequences, which flank the deleted regions (Edelman L et al., 1999). The name of this genetic disorders derives from Angelo DiGeorge who, in the late ‘60s, described this syndrome characterized by aberrant development of the thymus and parathyroid. Some of the development anomalies of DGS were also reproduced on chick models of neural crest ablation (Farrel MJ et al., 1999) leading investigators to hypothesize that DGS may derive from abnormal development of neural crest cell-contributed organs. However, more recent studies have implicated other cell lineages, specifically the cardiopharyngeal mesoderm, that will be discussed later. DGS is caused by chromosomal microdeletion of chromosome 22, at q11.2 locus; therefore, the disease is now commonly referred to as 22q11.2 deletion syndrome. Most patients with this syndrome have a large (3Mb) genomic deletion. About 10% of patients have a smaller deletion of about 1.5 Mb. Most genes localized in the region are conserved in the mouse on chromosome 16 (Gong W. et al., 1996; Botta A et al., 1997; Sutherland HF et al., 1998; Puech A et al., 1997; Lund J et al., 1999). This system allowed for the engineering of the first model, named Df1, which carries a deletion that encompasses mouse homologues of 18 genes that are deleted in patients with 1.5 Mb deletion whose phenotype is characterized by heart defects, thymus and parathyroid defects (Linsday EA et al., 1999). In particular, the cardiac defects may be rescued in Df1 mice on chromosome 16 by reciprocal duplication (Dp1) on the homolog, restoring normal dosage of the Df1 region.
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The role of routing policies in the internet : stability, security and load-balancing

The role of routing policies in the internet : stability, security and load-balancing

In Chapter 3, we continue our investigation on (Question 1), providing the main insight into BGP intractability. We unveil an intriguing analogy between logic circuits and BGP network configurations that leads to a surprising result: “BGP is Turing-Complete”. Put another way, analyzing BGP routing config- urations is as hard as debugging any computer program, which in most cases is an unfeasible feat. To achieve this result, it suffices to show how elementary BGP configurations can replicate the behavior of AND, OR, and NOT gates. In particular, the logic signals 1 and 0 are mapped to the absence or presence of a BGP route, respectively. Two elements are nonetheless missing to build a Turing machine: the ability to store information in the BGP configuration (i.e., a memory) and a clock. These elements are built by exploiting two pe- culiar real-world configurations that have troubled network operators and the IETF community for over a decade, showing that some real-world BGP con- figurations are capable of storing information in a very similar way electronic flip-flops do. BGP is therefore powerful enough to encode logic circuits of arbi- trary complexity and, as such, the BGP routing system constitutes the “biggest computer ” ever made!
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New perspectives in the diagnosis of cardiac allograft vasculopathy: the CT-scan role in the follow-up of heart transplanted patients

New perspectives in the diagnosis of cardiac allograft vasculopathy: the CT-scan role in the follow-up of heart transplanted patients

o Shumway technique (BIATRIAL technique): During implantation, perfusate temperature is generally28°C, with intermittent topical cooling using 4°C saline ice slush. No additional cardioplegic solution is infused. The left atrial anastomosis is constructed first using continuous 3-0 polypropylene suture When constructing it, he first few stitches are placed “at a distance” before lowering the donor heart into the pericardial space. The remainder of the entire left atrial anastomosis is constructed in an everting fashion to provide endothelium-to-endothelium apposition, thereby reducing the chance of thrombus formation along the suture line. Construction of the far-leftward portion of the anastomosis along the left pulmonary veins is often facilitated by retracting the donor ascending aorta inferiorly with a traction suture. The right atrial anastomosis is also constructed with continuous 3-0 polypropylene suture. In the area over the interatrial septum, the suture lines are partially overlapping. Each chamber is filled with cold saline before securing the suture lines. The aortic anastomosis is constructed with continuous 4-0 polypropylene suture after the donor and recipient aortas are cut to appropriate length. A cardioplegia catheter to be used as a “needle vent” for aspirating air is placed in the donor ascending aorta. Air is evacuated from the heart through the aortic suture line, and the suture line secured. The aortic clamp is removed with strong suction on the needle vent. When a gentle sinus rhythm is established, preparations are made for the pulmonary artery anastomosis. (Some surgeons prefer to complete this anastomosis before removing the aortic clamp.) The pulmonary artery segments are cut to an appropriate length and the anastomosis constructed, usually with 4-0 or 5-0 polypropylene suture. The remainder of the operation is conducted as usual during rewarming, and CPB is gradually discontinued after thoroughly de-airing the heart through the aortic needle vent while examining it for residual air with TEE.
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Importance of environmental habitats as a reservoir of phytopathogenic bacteria and their role in the evolution of pathogenicity traits

Importance of environmental habitats as a reservoir of phytopathogenic bacteria and their role in the evolution of pathogenicity traits

P. syringae phylogroup1. This phylogroup contains many strains from diseased plants but also from numerous environmental habitats and substrates [2] including precipitation, fresh water, epilithic biofilms and leaf litter (Table S1). Phylogroup 1 consists of two clades described by other authors [8,11]. Strains in clade 1a (including P. s. pv. tomato) gave variable results for phenotypic tests but all produced fluorescent pigment on KB medium and degraded esculin (Table 2). Clade 1b includes P. avellanae and P. s. pv. actinidiae, respectively the causal agents of bacterial canker of hazelnut and kiwifruit and also numerous strains isolated from snow, water and leaf litter (Table S1, S2). Strains in this clade, as well as strains in phylogroup 3, contain a catechol operon regrouping genes for degradation of aromatic compounds (unpublished data). Strains in clades 1a and 1b were similar in terms of their phenotypic variability, except that ca. 32 % of the latter did not produce fluorescent pigment on KB and 17 % did not degrade esculin. All the strains that did not degrade esculin carried the genes for degradation of aromatic compounds (unpublished data). Genomic studies have shown that among all phylogroups, strains of phylogroup 1 have the greatest number of Type Three Effector (T3E) genes coding for virulence determinants [25, 32]. More recently, Monteil and coworkers [33] demonstrated that strains closely related to the tomato speck pathogen P. s. pv. tomato isolated from snowpack and streams harbor the T3E genes found in epidemic strains. Most T3E gene expressions are driven by the HrpL sigma factor that also regulates non-T3E genes associated with virulence. All the genes regulated by the HrpL sigma factor are called HrpL regulons [34]. Consistent with this observation, the clade 1a strain P. s. pv. tomato DC3000 has the greatest number of HrpL regulons described to date [34].
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Investigating the role of Werner syndrome protein in the activation of the ATR-dependent checkpoint in response to mild replication stress

Investigating the role of Werner syndrome protein in the activation of the ATR-dependent checkpoint in response to mild replication stress

Cells from WS individuals have a short replicative lifespan in culture WS, in fact cells undergo highly premature replicative senescence, failing to proliferate after only 9-11 population doublings, compared with the 50-60 doublings characteristic of wild type fibroblasts (Hayflick, 1979). Transcriptomic studies have demonstrated that gene expression profiling in Werner syndrome closely resembles those of normal aging, with >90% gene expression changes associated with normal ageing seen in young WS cells (Kyng et al., 2003).Moreover, WS cells exhibit genomic instability characterized by chromosomal variegated translocation mosaicism (Salk et al., 1985), and more in general spontaneous chromosomal abnormalities and large deletions in many genes(Fukuchi et al., 1989; Gowans et al., 2005), which may represent an important determinant of the increased risk of cancer and of the aging phenotype (van Brabant et al., 2000; Goto, 1997).WS cells show phenotypes such as non-homologous chromosome exchanges and large chromosomal deletions, caused by deficiency of DSBR (Singh et al., 2009). WRN can also catalyse branch migration of Holliday junctions and melting of D-loops, which represent recombination intermediates. Moreover, it has been established that WRN participates in a multi-protein complex including ATR and the recombination proteins RAD51, RAD52, RAD54 and RAD54B, supporting a role for WRN in the later steps of the HR process (Otterlei et al., 2006). WS cells are sensitive to hydrogen peroxide (Von Kobbe et al., 2004), supporting, together with biochemical evidence (Harrigan et al., 2006), the involvement of WRN in base excision repair BER, that is one of the major DNA repair pathways next nucleotide excision repair (NER), double strand break repair (DSBR), and mismatch repair (MMR).WS cells are very sensitive to a well known DSB generating agent. Rapid accumulation of WRN at laser-induced DSBs has been shown, and it remains at the DSB site for at least for 4 h (Singh et al., 2009). DSBs are repaired by either non-homologous end joining (NHEJ) or homologous recombination (HR) processes. WRN is known to physically and functionally interact with two key proteins involved in NHEJ, Ku and DNA-PKcs(Chen et al., 2003; Cooper et al., 2000; Karmakar, 2002).
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Analysis of the role of werner helicase interacting protein 1 in response to replication stress

Analysis of the role of werner helicase interacting protein 1 in response to replication stress

As described above, multiple pathways work in the restarting of stalled replication forks to allow DNA replication completion. Beyond the importance of restarting stalled forks, replication fork protection seems to be equally important to assure genomic stability, as it is underscored by the increasing number of proteins identified as being part of this process. Recent studies have underlined a crucial role for proteins involved in the Fanconi Anaemia (FA)/homologous recombination (HR) pathway in maintaining genome stability during replication stress (Costanzo, 2011). Components of this pathway have traditionally been associated with the HR-dependent repair of inter-strand crosslinks (ICLs), and mutations in these genes give rise to Fanconi Anaemia, a rare human disorder characterized by severe developmental abnormalities and tumor predisposition (Lord and Ashworth, 2007; Wang and Gautier, 2010). In addition to their importance in promoting the repair of ICLs, it is now apparent that several FA/HR proteins also play a role in protection and stabilization of stalled replication forks from uncontrolled nucleolysis (Hashimoto et al., 2010; Schlacher et al., 2011, 2012). If left unprotected, excessive nucleolytic processing (fork degradation) renders such forks unrecoverable, and may perturb replication to such an extent that stretches of under-replicated DNA accumulate. Therefore, these fork protection factors represent important barriers to prevent genome instability.
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Regulation of quorum sensing and virulence in pseudomonas aeruginosa

Regulation of quorum sensing and virulence in pseudomonas aeruginosa

From a physiological perspective, the relevance of this work is the demonstration that P. aeruginosa has the ability to modulate with different dynamics distinct subsets of LasR-controlled genes. In other terms, by altering the levels of a single intracellular receptor, LasR, P. aeruginosa can modulate the expression level of only the subset of the LasR-controlled genes that are not simultaneously regulate by RsaL. This complex regulatory behaviour likely enhances P. aeruginosa phenotypic plasticity, and might be relevant also during the infection process. P. aeruginosa can establish both acute and chronic infections, characterised by different behaviours and distinctive phenotypes. An acute infection is rapid, systemic and carried out by a planktonic bacterial community expressing high levels of virulence factors. Conversely, in a chronic infection bacterial proliferation is limited to a specific host tissue such as the lung of cystic fibrosis patients, and bacteria can persist in the host for extended periods of time, forming biofilm and adopting a slow- growing sessile lifestyle [50]. Despite the different patterns of phenotypes expressed by P. aeruginosa during the acute and the chronic infections, both are positively controlled by QS. Our results suggest that the different response of the two LasR sub-regulons to external cues affecting LasR may be involved in the different levels of expression of the virulence-related genes required in the acute and in the chronic infection. Indeed some LasR-controlled virulence factors, such as elastase and proteases, are largely expressed during acute infections, while their expression level is reduced during chronic infections [50]. Conversely, other LasR-controlled virulence factors, such as pyocyanin, are expressed in both the acute and chronic infections [28]. Notably, high levels of 3OC 12 -HSL and pyocyanin have been recovered in the sputum of cystic fibrosis patients with P.
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Study of the role of products and enzymes of cholesterol biosynthetic pathway in muscle tissues

Study of the role of products and enzymes of cholesterol biosynthetic pathway in muscle tissues

Fixed EDL muscles were dehydrated in graded ethanol, transferred to Bioclear (BioOptica, Milan, Italy) and then to a 1:1 mixture of Bioclear and paraffin, and finally embedded in paraffin. Transverse, 7-␮m-thick sections were cut by a microtome and collected on Vectabond precoated slides (Vector, Burlingame, CA, USA). Sections were deparaf- finized, rehydrated, and submitted to antigen retrieval, using antigen unmasking solution (Vector). After cooling, slides were transferred to PBS containing 3% hydrogen peroxide, for 5 min, in the dark, then to PBS with 0.2% Triton X-100 and 3% bovine serum albumin (BSA), for 1 h at room temperature. Sections were incubated overnight at 4°C with either of the following mouse polyclonal antibodies, diluted in PBS containing 0.1% Triton X-100 and 1.5% BSA: fast MHC MY-32 1:1000, slow MHC NOQ7.5.4D 1:5000 (Sigma). In control sections, the primary antibody was omitted. Slides were then incubated for 1 h at room temperature with biotinylated goat anti-rabbit IgG (Vector), diluted 1:200 in PBS containing 1% normal goat serum (Vector). Immuno- complexes were revealed by means of an avidin biotin system (Vectastain Elite ABC kit, Vector), using 3,3⬘-diamino-benzi- dine (DAB Substrate kit for peroxidase; Vector), as the chromogen. Sections were counterstained with hematoxylin, then dehydrated and mounted with Eukitt (Kindler GmbH & Co., Freiburg, Germany). Slides were observed under an Olympus BX 51 microscope (Olympus, Tokyo, Japan) equipped with a Leica DFC 420 camera (Leica Microsystems, Wetzlar, Germany); electronic images were captured by a Leica Application Suite system, and composed in an Adobe Photoshop CS2 format (Adobe Systems, San Jose, CA, USA).
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