THE SUBTLE BIOFILM REGULATION IN ESCHERICHIA COLI: CSGD AND THE YDDV-DOS OPERON

In this PhD thesis work I investigated the expression modulation of the major adhesion factors in Escherichia coli; in particular I focused on the role of GGDEF and EAL proteins, on their modulation in E. coli biofilm formation in response to environmental signals and on regulation of curli fibers, cellulose and poly-N-acetylglucosamine (PNAG), the most important biofilm determinants in E. coli. E. coli is an Enterobacterium, normally living inside the mammalian gut, at temperature of 37\ub0 C and in relatively nutrient-rich environment. Once outside the host, bacteria usually face much lower temperatures (< 30\ub0C) and a nutrient-limiting environment. The biofilm determinants studied in this thesis are all expressed in response to environmental conditions such as low temperature, low osmolarity and starvation, suggesting that E. coli bacteria switch to a biofilm mode of growth as part of their adaptation to the natural environment. In response to reduction in growth rates, E. coli seems to canalize its energy consumption into production of extracellular features such as curli or exopolysaccharides. Biofilms can be thus considered as a \u201cresistance form\u201d of growth able to withstand stress conditions more efficiently than cells living in a planktonic mode of growth. The CsgD protein is the master regulator of E. coli biofilm formation. It is a transcriptional factor necessary for curli genes transcription and, through the AdrA protein, for cellulose biosynthesis. Gene regulation by CsgD is tightly connected to production and sensing of cyclic di-GMP, a bacterial second messenger involved in various cellular processes, including biosynthesis of extracellular polysaccharides (Simm et al., 2004), biofilm formation (Hickman et al., 2005), and virulence (Pratt et al., 2007; Tischler and Camilli, 2005), as well as morphological and physiological differentiation (Paul et al., 2004). The CsgD-dependent adrA gene, involved in cellulose biosynthesis (Zogaj et al., 2001), encodes a cyclic di-GMP synthase (Simm et al., 2004). CsgD can also activate yoaD, whose gene product is a cyclic di-GMP phosphodiesterase, suggesting that CsgD is directly involved in feedback regulation of cyclic di-GMP intracellular levels and of cellulose biosynthesis (Brombacher et al., 2006). CsgD is also able to activate the iraP gene: IraP acts as a stabilization factor for the \u3c3s protein, an alternative sigma factor of RNA polymerase which directs transcription of genes involved in adaptation to slow growth and to cellular stresses. Here I showed that CsgD transcription activation of the iraP gene does result in a significant increase of \u3c3s intracellular concentration by positively affecting \u3c3s protein stability, thus leading to altered expression of \u3c3s-dependent genes. CsgD-mediated increase of \u3c3s cellular concentrations via the iraP gene would trigger an autoactivation loop leading to an increased production of CsgD-dependent adhesion determinants such as curli fibers and cellulose. This autoregulatory circuitry might be further fueled by \u3c3s-dependent induction of genes encoding di-guanylate cyclases, i.e., proteins able to synthesize the second messenger di-cyclic- GMP, which, in turn, can positively affect csg gene expression (Kader et al., 2006; Weber et al., 2002). The yddV-dos operon is the most expressed among c-di-GMP-related genes showing dependence on \u3c3s (Weber et al., 2006; Sommerfeldt et al., 2009). It encodes, respectively, a protein with DGC activity and a PDE that can degrade c-di- GMP to pGpG. Both Dos and YddV are heme-binding oxygen sensors, and interact to form a stable protein complex (Tuckerman et al., 2009). Although it has been reported that YddV overexpression can stimulate biofilm formation (Mendez-Ortiz et al., 2006), the targets of yddV-dependent biofilm induction had not yet been identified. Here I showed that YddV acts modulating curli and PNAG expression. Control of curli production by yddV-dos takes place at the level of transcription regulation of the csgBAC operon, encoding curli structural subunits, and is mediated by the DGC and PDE activities of YddV and Dos. In contrast, the YddV\u2013Dos protein complex does not strongly influence csgDEFG expression, nor does it affect the expression of the CsgD-dependent adrA gene, encoding a positive effector for cellulose biosynthesis. Regarding PNAG production, we showed that YddV is able to prevent degradation of pgaABCD transcript in the MG1655csrA background, thus suggesting that a DGC might regulate gene expression by affecting mRNA stability in E. coli. YddV regulation of pgaABCD operon in a wild type contest is still controversial: pgaABCD genes are expressed at low levels in MG1655 (the standard laboratory strain of E. coli) and their mRNA half-life is lower than two minutes regardless of the growth conditions tested; thus, possible effects of yddV inactivation on destabilization of the pga transcript are not easy to evaluate in the wt contest. In the last part of my thesis I tried to characterize a biofilm-forming mutant of E. coli, able to express pgaABCD genes at high levels. Even if initial data suggested that a mutation in the csrA gene could be responsible for pga mRNA stabilization in this mutant, actual the mutation leading to the adhesive phenotype and to PNAG production is outside the csrA gene and is still unknown. Moreover my data suggest a connection between pga expression and iron regulation in E. coli strains: it is conceivable that pgaABCD expression and consequent biofilm formation and the adherent phenotype depends on concerted production of different determinants, whose expression is also affected by iron concentration. Thus, my research highlighted that biofilm production is the result of coordinated expression of different adhesion determinants, whose regulation is complex and not fully understood. In particular, the precise extent and the molecular mechanism of c-di-GMP adhesion factors regulation remains to be largely identified and represents an exciting challenge for future research in the biofilm field

Ocean biogeochemistry exhibits contrasting responses to a large scale reduction in dust deposition

Dust deposition of iron is thought to be an important control on ocean biogeochemistry and air-sea CO&lt;sub&gt;2&lt;/sub&gt; exchange. In this study, we examine the impact of a large scale, yet climatically realistic, reduction in the aeolian Fe input during a 240 year transient simulation. In contrast to previous studies, we find that the ocean biogeochemical cycles of carbon and nitrogen are relatively insensitive (globally) to a 60% reduction in Fe input from dust. Net primary productivity (NPP) is reduced in the Fe limited regions, but the excess macronutrients that result are able to fuel additional NPP elsewhere. Overall, NPP and air-sea CO&lt;sub&gt;2&lt;/sub&gt; exchange are only reduced by around 3% between 1860 and 2100. While the nitrogen cycle is perturbed more significantly (by ~15%), reduced N&lt;sub&gt;2&lt;/sub&gt; fixation is balanced by a concomitant decline in denitrification. Feedbacks between N&lt;sub&gt;2&lt;/sub&gt; fixation and denitrification are controlled by variability in surface utilization of inorganic nitrogen and subsurface oxygen consumption, as well as the direct influence of Fe on N&lt;sub&gt;2&lt;/sub&gt; fixation. Overall, there is relatively little impact of reduced aeolian Fe input (&amp;lt;4%) on cumulative CO&lt;sub&gt;2&lt;/sub&gt; fluxes over 240 years. The lower sensitivity of our model to changes in dust input is primarily due to the more detailed representation of the continental shelf Fe, which was absent in previous models

Life Cycle Assessment through BIM-based advanced Calculation Virtual Environment workflows

The current built stock in Europe is large and energetically inefficient due to material decay caused by lifelong use and lack of maintenance, combined. New buildings are commissioned and delivered everyday with an environmental cost, calculated using the Life Cycle Assessment methodology. The LCA is now widely requested for new assets, while existing ones are left for demolition and consequent carbon emissions released into the atmosphere, increasing the environmental crisis. In an effort to increase built stock's energy efficiency rates and decrease carbon emission, the recovery of these assets is depicted as the strategic next step for the construction sector. Both goals are achieved using data generated by dedicated construction software and processed in Calculation Virtual Environment, according to the specific needed data. The use of BIM for refurbishment design projects allows virtual construction separated by phases to simulate existing conditions of the envelope and its improvement with insulation material options. The use of one single file with clear phase definitions ensures data extraction and transference for all material options. The process is not entirely automated nevertheless it allows to save, use and share data whenever needed. The connection between data sources and databases in the cloud provides timesaving and regular updates. The research findings demonstrate the positive outcomes of modeling existing structures for energy simulation oriented to use in LCA, increasing the potential of BIM use in sensitive constructions while delivering appropriate results based on model and enriched geometry, with cost evaluation potential enabling scenario comparison for better decision making

100 Years of BCG Immunization: Past, Present, and Future

The 100th anniversary of the introduction of Bacille&ndash;Calmette&ndash;Gu&eacute;rin (BCG) as a tuberculosis (TB) vaccine is an occasion warranting further investigation of the early attempts which culminated in the introduction of BCG as a TB vaccine, as well as of subsequent recognition of failures, new findings that broaden its applications, outstanding questions, and approaches towards the development of novel vaccine candidates [...

SCAN-TO-BIM EFFICIENT APPROACH TO EXTRACT BIM MODELS FROM HIGH PRODUCTIVE INDOOR MOBILE MAPPING SURVEY

Building Information Modeling represents one of the most interesting developments in construction fields in the last 20 years. BIM process supports the creation of intelligent data that can be used throughout the life cycle of a construction project. Where a project involves a pre-existing structure, reality capture can provide the most critical information. The purpose of this paper is to describe an efficient approach to extract 3D models using high productive indoor Mobile Mapping Systems (iMMS) and an optimized scan-to-BIM workflow. The scan-to-BIM procedure allows reconstructing several elements within a digital environment preserving the features and reusing them in the development of the BIM project. The elaboration of the raw data acquired from the iMMS starts with the software HERON® Desktop where a SLAM algorithm runs and a 3D point cloud model is produced. The model is translated in the Gexcel Reconstructor® point cloud post processing software where a number of deliverables as orthophotos, blueprints and a filtered and optimized point cloud are obtained. In the proposed processing workflow, the data are introduced to Autodesk ReCap®, where the model can be edited and the final texturized point cloud model extracted. The identification and modeling of the 3D objects that compose the BIM model is realized in ClearEdge3D EdgeWiseTM and optimized in Autodesk Revit®. The data elaboration workflow implemented shows how an optimized data processing workflow allows making the scan-to-BIM procedure automatic and economically sustainable

Behavioural Differences in Sensorimotor Profiles: A Comparison of Preschool-Aged Children with Sensory Processing Disorder and Autism Spectrum Disorders

Sensory Processing Disorders (SPDs) define dysfunctions in modulating, organising, and using information from several sensory channels for regulating motor, behavioural, emotional and attention responses. Although SPD can be identified also as an isolated condition in young children, its presence in Autism Spectrum Disorder (ASD) population is really frequent. The study purpose is to explore the SPD clinical expression and the putative correlation with several behavioural aspects both in children with ASD and in those with isolated SPD. Therefore, 43 preschool-aged children (25 ASD vs. 18 SPD) were recruited, and their parents completed three questionnaires (Developmental Profile-3, Sensory Processing Measure–Preschool, Repetitive Behaviour Scale-Revised) to evaluate behavioural alterations and developmental levels. The main result is that both ASD and SPD groups had significantly sensory-related behavioural symptoms, although ASD children seem to be more impaired in all areas. Several significant correlations were found between sensory processing difficulties and repetitive behaviours, but in the SPD group a specific relationship between Body Awareness and Ritualistic/Sameness Behaviour was found. Conversely, in the ASD group, more diffuse interlinks between sensory processing difficulties and motor behaviours were significant. In conclusion, the present study confirms the key role of sensory–motor skills in early diagnosis and intervention among children at risk for neurodevelopmental disorders