Regulatory networks of Neisseria meningitidis and their implications for pathogenesis

Neisseria meningitidis, the leading cause of bacterial meningitis, can adapt to different host niches during human infection. Both transcriptional and post-transcriptional regulatory networks have been identified as playing a crucial role for bacterial stress responses and virulence. We investigated the N. meningitidis transcriptional landscape both by microarray and by RNA sequencing (RNAseq). Microarray analysis of N. meningitidis grown in the presence or absence of glucose allowed us to identify genes regulated by carbon source availability. In particular, we identified a glucose-responsive hexR-like transcriptional regulator in N. meningitidis. Deletion analysis showed that the hexR gene is accountable for a subset of the glucose-responsive regulation, and in vitro assays with the purified protein showed that HexR binds to the promoters of the central metabolic operons of meningococcus, by targeting a DNA region overlapping putative regulatory sequences. Our results indicate that HexR coordinates the central metabolism of meningococcus in response to the availability of glucose, and N. meningitidis strains lacking the hexR gene are also deficient in establishing successful bacteremia in a mouse model of infection. In parallel, RNAseq analysis of N. meningitidis cultured under standard or iron-limiting in vitro growth conditions allowed us to identify novel small non-coding RNAs (sRNAs) potentially involved in N. meningitidis regulatory networks. Manual curation of the RNAseq data generated a list of 51 sRNAs, 8 of which were validated by Northern blotting. Deletion of selected sRNAs caused attenuation of N. meningitidis infection in a murine model, leading to the identification of the first sRNAs influencing meningococcal bacteraemia. Furthermore, we describe the identification and initial characterization of a novel sRNA unique to meningococcus, closely associated to genes relevant for the intracellular survival of pathogenic Neisseriae. Taken together, our findings could help unravel the regulation of N. meningitidis adaptation to the host environment and its implications for pathogenesis.

Rapporti fra fattori ambientali e proteine di parete in Bifidobacterium

The normal gut microbiota has several important functions in host physiology and metabolism, and plays a key role in health and disease. Bifidobacteria, which are indigenous components of gastrointestinal microbiota, may play an important role in maintaining the well-being of the host although its precise function is very difficult to study. Its physiological and biochemical activities are controlled by many factors, particularly diet and environment. Adherence and colonization capacity are considered as contributing factors for immune modulation, pathogen exclusion, and enhanced contact with the mucosa. In this way, bifidobacteria would fortify the microbiota that forms an integral part of the mucosal barrier and colonization resistance against pathogens. Bifidobacteria are not only subjected to stressful conditions in industrial processes, but also in nature, where the ability to respond quickly to stress is essential for survival. Bifidobacteria, like other microorganisms, have evolved sensing systems for/and defences against stress that allow them to withstand harsh conditions and sudden environmental changes. Bacterial stress responses rely on the coordinated expression of genes that alter various cellular processes and structures (e.g. DNA metabolism, housekeeping genes, cell-wall proteins, membrane composition) and act in concert to improve bacterial stress tolerance. The integration of these stress responses is accomplished by regulatory networks that allow the cell to react rapidly to various and sometimes complex environmental changes. This work examined the effect of important stressful conditions, such as changing pH and osmolarity, on the biosynthesis of cell wall proteins in B. pseudolongum subsp. globosum. These environmental factors all influence heavily the expression of BIFOP (BIFidobacterial Outer Proteins) in the cell-wall and can have an impact in the interaction with host. Also evidence has been collected linking the low concentration of sugar in the culture medium with the presence or absence of extracromosomal DNA.

Identification and characterization of the sRNA network in Neisseria meningitidis

Bacterial small regulatory RNAs (sRNAs) are posttranscriptional regulators involved in stress responses. These short non-coding transcripts are synthesised in response to a signal, and control gene expression of their regulons by modulating the translation or stability of the target mRNAs, often in concert with the RNA chaperone Hfq. Characterization of a Hfq knock out mutant in Neisseria meningitidis revealed that it has a pleiotropic phenotype, suggesting a major role for Hfq in adaptation to stresses and virulence and the presence of Hfq-dependent sRNA activity. Global gene expression analysis of regulated transcripts in the Hfq mutant revealed the presence of a regulated sRNA, incorrectly annotated as an open reading frame, which we renamed AniS. The synthesis of this novel sRNA is anaerobically induced through activation of its promoter by the FNR global regulator and through global gene expression analyses we identified at least two predicted mRNA targets of AniS. We also performed a detailed molecular analysis of the action of the sRNA NrrF,. We demonstrated that NrrF regulates succinate dehydrogenase by forming a duplex with a region of complementarity within the sdhDA region of the succinate dehydrogenase transcript, and Hfq enhances the binding of this sRNA to the identified target in the sdhCDAB mRNA; this is likely to result in rapid turnover of the transcript in vivo. In addition, in order to globally investigate other possible sRNAs of N. meningitdis we Deep-sequenced the transcriptome of this bacterium under both standard in vitro and iron-depleted conditions. This analysis revealed genes that were actively transcribed under the two conditions. We focused our attention on the transcribed non-coding regions of the genome and, along with 5’ and 3’ untranslated regions, 19 novel candidate sRNAs were identified. Further studies will be focused on the identification of the regulatory networks of these sRNAs, and their targets.

Metaheuristics for Search Problems in Genomics - New Algorithms and Applications

In this thesis we made the first steps towards the systematic application of a methodology for automatically building formal models of complex biological systems. Such a methodology could be useful also to design artificial systems possessing desirable properties such as robustness and evolvability. The approach we follow in this thesis is to manipulate formal models by means of adaptive search methods called metaheuristics. In the first part of the thesis we develop state-of-the-art hybrid metaheuristic algorithms to tackle two important problems in genomics, namely, the Haplotype Inference by parsimony and the Founder Sequence Reconstruction Problem. We compare our algorithms with other effective techniques in the literature, we show strength and limitations of our approaches to various problem formulations and, finally, we propose further enhancements that could possibly improve the performance of our algorithms and widen their applicability. In the second part, we concentrate on Boolean network (BN) models of gene regulatory networks (GRNs). We detail our automatic design methodology and apply it to four use cases which correspond to different design criteria and address some limitations of GRN modeling by BNs. Finally, we tackle the Density Classification Problem with the aim of showing the learning capabilities of BNs. Experimental evaluation of this methodology shows its efficacy in producing network that meet our design criteria. Our results, coherently to what has been found in other works, also suggest that networks manipulated by a search process exhibit a mixture of characteristics typical of different dynamical regimes.

Master Equation: Biological Applications and Thermodynamic Description

It is well known that many realistic mathematical models of biological systems, such as cell growth, cellular development and differentiation, gene expression, gene regulatory networks, enzyme cascades, synaptic plasticity, aging and population growth need to include stochasticity. These systems are not isolated, but rather subject to intrinsic and extrinsic fluctuations, which leads to a quasi equilibrium state (homeostasis). The natural framework is provided by Markov processes and the Master equation (ME) describes the temporal evolution of the probability of each state, specified by the number of units of each species. The ME is a relevant tool for modeling realistic biological systems and allow also to explore the behavior of open systems. These systems may exhibit not only the classical thermodynamic equilibrium states but also the nonequilibrium steady states (NESS). This thesis deals with biological problems that can be treat with the Master equation and also with its thermodynamic consequences. It is organized into six chapters with four new scientific works, which are grouped in two parts: (1) Biological applications of the Master equation: deals with the stochastic properties of a toggle switch, involving a protein compound and a miRNA cluster, known to control the eukaryotic cell cycle and possibly involved in oncogenesis and with the propose of a one parameter family of master equations for the evolution of a population having the logistic equation as mean field limit. (2) Nonequilibrium thermodynamics in terms of the Master equation: where we study the dynamical role of chemical fluxes that characterize the NESS of a chemical network and we propose a one parameter parametrization of BCM learning, that was originally proposed to describe plasticity processes, to study the differences between systems in DB and NESS.

Development and characterization of a distributed measurement system for the evaluation of voltage quality in electric power networks

The research activity carried out during the PhD course in Electrical Engineering belongs to the branch of electric and electronic measurements. The main subject of the present thesis is a distributed measurement system to be installed in Medium Voltage power networks, as well as the method developed to analyze data acquired by the measurement system itself and to monitor power quality. In chapter 2 the increasing interest towards power quality in electrical systems is illustrated, by reporting the international research activity inherent to the problem and the relevant standards and guidelines emitted. The aspect of the quality of voltage provided by utilities and influenced by customers in the various points of a network came out only in recent years, in particular as a consequence of the energy market liberalization. Usually, the concept of quality of the delivered energy has been associated mostly to its continuity. Hence the reliability was the main characteristic to be ensured for power systems. Nowadays, the number and duration of interruptions are the “quality indicators” commonly perceived by most customers; for this reason, a short section is dedicated also to network reliability and its regulation. In this contest it should be noted that although the measurement system developed during the research activity belongs to the field of power quality evaluation systems, the information registered in real time by its remote stations can be used to improve the system reliability too. Given the vast scenario of power quality degrading phenomena that usually can occur in distribution networks, the study has been focused on electromagnetic transients affecting line voltages. The outcome of such a study has been the design and realization of a distributed measurement system which continuously monitor the phase signals in different points of a network, detect the occurrence of transients superposed to the fundamental steady state component and register the time of occurrence of such events. The data set is finally used to locate the source of the transient disturbance propagating along the network lines. Most of the oscillatory transients affecting line voltages are due to faults occurring in any point of the distribution system and have to be seen before protection equipment intervention. An important conclusion is that the method can improve the monitored network reliability, since the knowledge of the location of a fault allows the energy manager to reduce as much as possible both the area of the network to be disconnected for protection purposes and the time spent by technical staff to recover the abnormal condition and/or the damage. The part of the thesis presenting the results of such a study and activity is structured as follows: chapter 3 deals with the propagation of electromagnetic transients in power systems by defining characteristics and causes of the phenomena and briefly reporting the theory and approaches used to study transients propagation. Then the state of the art concerning methods to detect and locate faults in distribution networks is presented. Finally the attention is paid on the particular technique adopted for the same purpose during the thesis, and the methods developed on the basis of such approach. Chapter 4 reports the configuration of the distribution networks on which the fault location method has been applied by means of simulations as well as the results obtained case by case. In this way the performance featured by the location procedure firstly in ideal then in realistic operating conditions are tested. In chapter 5 the measurement system designed to implement the transients detection and fault location method is presented. The hardware belonging to the measurement chain of every acquisition channel in remote stations is described. Then, the global measurement system is characterized by considering the non ideal aspects of each device that can concur to the final combined uncertainty on the estimated position of the fault in the network under test. Finally, such parameter is computed according to the Guide to the Expression of Uncertainty in Measurements, by means of a numeric procedure. In the last chapter a device is described that has been designed and realized during the PhD activity aiming at substituting the commercial capacitive voltage divider belonging to the conditioning block of the measurement chain. Such a study has been carried out aiming at providing an alternative to the used transducer that could feature equivalent performance and lower cost. In this way, the economical impact of the investment associated to the whole measurement system would be significantly reduced, making the method application much more feasible.

Planning and flexible operation of storage systems in power grids: from transmission to distribution networks

The first part of the thesis has been devoted to the transmission planning with high penetration of renewable energy sources. Both stationary and transportable battery energy storage (BES, BEST) systems have been considered in the planning model, so to obtain the optimal set of BES, BEST and transmission lines that minimizes the total cost in a power network. First, a coordinated expansion planning model with fixed transportation cost for BEST devices has been presented; then, the model has been extended to a planning formulation with a distance-dependent transportation cost for the BEST units, and its tractability has been proved through a case study based on a 190-bus test system. The second part of this thesis is then devoted to the analysis of planning and management of renewable energy communities (RECs). Initially, the planning of photovoltaic and BES systems in a REC with an incentive-based remuneration scheme according to the Italian regulatory framework has been analysed, and two planning models, according to a single-stage, or a multi-stage approach, have been proposed in order to provide the optimal set of BES and PV systems allowing to achieve the minimum energy procurement cost in a given REC. Further, the second part of this thesis is devoted to the study of the day-ahead scheduling of resources in renewable energy communities, by considering two types of REC. The first one, which we will refer to as “cooperative community”, allows direct energy transactions between members of the REC; the second type of REC considered, which we shall refer to as “incentive-based”, does not allow direct transactions between members but includes economic revenues for the community shared energy, according to the Italian regulation framework. Moreover, dispatchable renewable energy generation has been considered by including producers equipped with biogas power plants in the community.