Identification of Drosophila heart-specific Cis-Regulatory Modules under Hox control

Cardiac morphogenesis is a complex process governed by evolutionarily conserved transcription factors and signaling molecules. The Drosophila cardiac tube is linear, made of 52 pairs of cardiomyocytes (CMs), which express specific transcription factor genes that have human homologues implicated in Congenital Heart Diseases (CHDs) (NKX2-5, GATA4 and TBX5). The Drosophila cardiac tube is linear and composed of a rostral portion named aorta and a caudal one called heart, distinguished by morphological and functional differences controlled by Hox genes, key regulators of axial patterning. Overexpression and inactivation of the Hox gene abdominal-A (abd-A), which is expressed exclusively in the heart, revealed that abd-A controls heart identity. The aim of our work is to isolate the heart-specific cisregulatory sequences of abd-A direct target genes, the realizator genes granting heart identity. In each segment of the heart, four pairs of cardiomyocytes (CMs) express tinman (tin), homologous to NKX2-5, and acquire strong contractile and automatic rhythmic activities. By tyramide amplified FISH, we found that seven genes, encoding ion channels, pumps or transporters, are specifically expressed in the Tin-CMs of the heart. We initially used online available tools to identify their heart-specific cisregutatory modules by looking for Conserved Non-coding Sequences containing clusters of binding sites for various cardiac transcription factors, including Hox proteins. Based on these data we generated several reporter gene constructs and transgenic embryos, but none of them showed reporter gene expression in the heart. In order to identify additional abd-A target genes, we performed microarray experiments comparing the transcriptomes of aorta versus heart and identified 144 genes overexpressed in the heart. In order to find the heart-specific cis-regulatory regions of these target genes we developed a new bioinformatic approach where prediction is based on pattern matching and ordered statistics. We first retrieved Conserved Noncoding Sequences from the alignment between the D.melanogaster and D.pseudobscura genomes. We scored for combinations of conserved occurrences of ABD-A, ABD-B, TIN, PNR, dMEF2, MADS box, T-box and E-box sites and we ranked these results based on two independent strategies. On one hand we ranked the putative cis-regulatory sequences according to best scored ABD-A biding sites, on the other hand we scored according to conservation of binding sites. We integrated and ranked again the two lists obtained independently to produce a final rank. We generated nGFP reporter construct flies for in vivo validation. We identified three 1kblong heart-specific enhancers. By in vivo and in vitro experiments we are determining whether they are direct abd-A targets, demonstrating the role of a Hox gene in the realization of heart identity. The identified abd-A direct target genes may be targets also of the NKX2-5, GATA4 and/or TBX5 homologues tin, pannier and Doc genes, respectively. The identification of sequences coregulated by a Hox protein and the homologues of transcription factors causing CHDs, will provide a mean to test whether these factors function as Hox cofactors granting cardiac specificity to Hox proteins, increasing our knowledge on the molecular mechanisms underlying CHDs. Finally, it may be investigated whether these Hox targets are involved in CHDs.

Seizure prediction and control in epilepsy

The first part of my thesis presents an overview of the different approaches used in the past two decades in the attempt to forecast epileptic seizure on the basis of intracranial and scalp EEG. Past research could reveal some value of linear and nonlinear algorithms to detect EEG features changing over different phases of the epileptic cycle. However, their exact value for seizure prediction, in terms of sensitivity and specificity, is still discussed and has to be evaluated. In particular, the monitored EEG features may fluctuate with the vigilance state and lead to false alarms. Recently, such a dependency on vigilance states has been reported for some seizure prediction methods, suggesting a reduced reliability. An additional factor limiting application and validation of most seizure-prediction techniques is their computational load. For the first time, the reliability of permutation entropy [PE] was verified in seizure prediction on scalp EEG data, contemporarily controlling for its dependency on different vigilance states. PE was recently introduced as an extremely fast and robust complexity measure for chaotic time series and thus suitable for online application even in portable systems. The capability of PE to distinguish between preictal and interictal state has been demonstrated using Receiver Operating Characteristics (ROC) analysis. Correlation analysis was used to assess dependency of PE on vigilance states. Scalp EEG-Data from two right temporal epileptic lobe (RTLE) patients and from one patient with right frontal lobe epilepsy were analysed. The last patient was included only in the correlation analysis, since no datasets including seizures have been available for him. The ROC analysis showed a good separability of interictal and preictal phases for both RTLE patients, suggesting that PE could be sensitive to EEG modifications, not visible on visual inspection, that might occur well in advance respect to the EEG and clinical onset of seizures. However, the simultaneous assessment of the changes in vigilance showed that: a) all seizures occurred in association with the transition of vigilance states; b) PE was sensitive in detecting different vigilance states, independently of seizure occurrences. Due to the limitations of the datasets, these results cannot rule out the capability of PE to detect preictal states. However, the good separability between pre- and interictal phases might depend exclusively on the coincidence of epileptic seizure onset with a transition from a state of low vigilance to a state of increased vigilance. The finding of a dependency of PE on vigilance state is an original finding, not reported in literature, and suggesting the possibility to classify vigilance states by means of PE in an authomatic and objectic way. The second part of my thesis provides the description of a novel behavioral task based on motor imagery skills, firstly introduced (Bruzzo et al. 2007), in order to study mental simulation of biological and non-biological movement in paranoid schizophrenics (PS). Immediately after the presentation of a real movement, participants had to imagine or re-enact the very same movement. By key release and key press respectively, participants had to indicate when they started and ended the mental simulation or the re-enactment, making it feasible to measure the duration of the simulated or re-enacted movements. The proportional error between duration of the re-enacted/simulated movement and the template movement were compared between different conditions, as well as between PS and healthy subjects. Results revealed a double dissociation between the mechanisms of mental simulation involved in biological and non-biologial movement simulation. While for PS were found large errors for simulation of biological movements, while being more acurate than healthy subjects during simulation of non-biological movements. Healthy subjects showed the opposite relationship, making errors during simulation of non-biological movements, but being most accurate during simulation of non-biological movements. However, the good timing precision during re-enactment of the movements in all conditions and in both groups of participants suggests that perception, memory and attention, as well as motor control processes were not affected. Based upon a long history of literature reporting the existence of psychotic episodes in epileptic patients, a longitudinal study, using a slightly modified behavioral paradigm, was carried out with two RTLE patients, one patient with idiopathic generalized epilepsy and one patient with extratemporal lobe epilepsy. Results provide strong evidence for a possibility to predict upcoming seizures in RTLE patients behaviorally. In the last part of the thesis it has been validated a behavioural strategy based on neurobiofeedback training, to voluntarily control seizures and to reduce there frequency. Three epileptic patients were included in this study. The biofeedback was based on monitoring of slow cortical potentials (SCPs) extracted online from scalp EEG. Patients were trained to produce positive shifts of SCPs. After a training phase patients were monitored for 6 months in order to validate the ability of the learned strategy to reduce seizure frequency. Two of the three refractory epileptic patients recruited for this study showed improvements in self-management and reduction of ictal episodes, even six months after the last training session.

Architectures and design patterns for functional design of logic control and diagnostics in industrial automation

Recently in most of the industrial automation process an ever increasing degree of automation has been observed. This increasing is motivated by the higher requirement of systems with great performance in terms of quality of products/services generated, productivity, efficiency and low costs in the design, realization and maintenance. This trend in the growth of complex automation systems is rapidly spreading over automated manufacturing systems (AMS), where the integration of the mechanical and electronic technology, typical of the Mechatronics, is merging with other technologies such as Informatics and the communication networks. An AMS is a very complex system that can be thought constituted by a set of flexible working stations, one or more transportation systems. To understand how this machine are important in our society let considerate that every day most of us use bottles of water or soda, buy product in box like food or cigarets and so on. Another important consideration from its complexity derive from the fact that the the consortium of machine producers has estimated around 350 types of manufacturing machine. A large number of manufacturing machine industry are presented in Italy and notably packaging machine industry,in particular a great concentration of this kind of industry is located in Bologna area; for this reason the Bologna area is called “packaging valley”. Usually, the various parts of the AMS interact among them in a concurrent and asynchronous way, and coordinate the parts of the machine to obtain a desiderated overall behaviour is an hard task. Often, this is the case in large scale systems, organized in a modular and distributed manner. Even if the success of a modern AMS from a functional and behavioural point of view is still to attribute to the design choices operated in the definition of the mechanical structure and electrical electronic architecture, the system that governs the control of the plant is becoming crucial, because of the large number of duties associated to it. Apart from the activity inherent to the automation of themachine cycles, the supervisory system is called to perform other main functions such as: emulating the behaviour of traditional mechanical members thus allowing a drastic constructive simplification of the machine and a crucial functional flexibility; dynamically adapting the control strategies according to the different productive needs and to the different operational scenarios; obtaining a high quality of the final product through the verification of the correctness of the processing; addressing the operator devoted to themachine to promptly and carefully take the actions devoted to establish or restore the optimal operating conditions; managing in real time information on diagnostics, as a support of the maintenance operations of the machine. The kind of facilities that designers can directly find on themarket, in terms of software component libraries provides in fact an adequate support as regard the implementation of either top-level or bottom-level functionalities, typically pertaining to the domains of user-friendly HMIs, closed-loop regulation and motion control, fieldbus-based interconnection of remote smart devices. What is still lacking is a reference framework comprising a comprehensive set of highly reusable logic control components that, focussing on the cross-cutting functionalities characterizing the automation domain, may help the designers in the process of modelling and structuring their applications according to the specific needs. Historically, the design and verification process for complex automated industrial systems is performed in empirical way, without a clear distinction between functional and technological-implementation concepts and without a systematic method to organically deal with the complete system. Traditionally, in the field of analog and digital control design and verification through formal and simulation tools have been adopted since a long time ago, at least for multivariable and/or nonlinear controllers for complex time-driven dynamics as in the fields of vehicles, aircrafts, robots, electric drives and complex power electronics equipments. Moving to the field of logic control, typical for industrial manufacturing automation, the design and verification process is approached in a completely different way, usually very “unstructured”. No clear distinction between functions and implementations, between functional architectures and technological architectures and platforms is considered. Probably this difference is due to the different “dynamical framework”of logic control with respect to analog/digital control. As a matter of facts, in logic control discrete-events dynamics replace time-driven dynamics; hence most of the formal and mathematical tools of analog/digital control cannot be directly migrated to logic control to enlighten the distinction between functions and implementations. In addition, in the common view of application technicians, logic control design is strictly connected to the adopted implementation technology (relays in the past, software nowadays), leading again to a deep confusion among functional view and technological view. In Industrial automation software engineering, concepts as modularity, encapsulation, composability and reusability are strongly emphasized and profitably realized in the so-calledobject-oriented methodologies. Industrial automation is receiving lately this approach, as testified by some IEC standards IEC 611313, IEC 61499 which have been considered in commercial products only recently. On the other hand, in the scientific and technical literature many contributions have been already proposed to establish a suitable modelling framework for industrial automation. During last years it was possible to note a considerable growth in the exploitation of innovative concepts and technologies from ICT world in industrial automation systems. For what concerns the logic control design, Model Based Design (MBD) is being imported in industrial automation from software engineering field. Another key-point in industrial automated systems is the growth of requirements in terms of availability, reliability and safety for technological systems. In other words, the control system should not only deal with the nominal behaviour, but should also deal with other important duties, such as diagnosis and faults isolations, recovery and safety management. Indeed, together with high performance, in complex systems fault occurrences increase. This is a consequence of the fact that, as it typically occurs in reliable mechatronic systems, in complex systems such as AMS, together with reliable mechanical elements, an increasing number of electronic devices are also present, that are more vulnerable by their own nature. The diagnosis problem and the faults isolation in a generic dynamical system consists in the design of an elaboration unit that, appropriately processing the inputs and outputs of the dynamical system, is also capable of detecting incipient faults on the plant devices, reconfiguring the control system so as to guarantee satisfactory performance. The designer should be able to formally verify the product, certifying that, in its final implementation, it will perform itsrequired function guarantying the desired level of reliability and safety; the next step is that of preventing faults and eventually reconfiguring the control system so that faults are tolerated. On this topic an important improvement to formal verification of logic control, fault diagnosis and fault tolerant control results derive from Discrete Event Systems theory. The aimof this work is to define a design pattern and a control architecture to help the designer of control logic in industrial automated systems. The work starts with a brief discussion on main characteristics and description of industrial automated systems on Chapter 1. In Chapter 2 a survey on the state of the software engineering paradigm applied to industrial automation is discussed. Chapter 3 presentes a architecture for industrial automated systems based on the new concept of Generalized Actuator showing its benefits, while in Chapter 4 this architecture is refined using a novel entity, the Generalized Device in order to have a better reusability and modularity of the control logic. In Chapter 5 a new approach will be present based on Discrete Event Systems for the problemof software formal verification and an active fault tolerant control architecture using online diagnostic. Finally conclusive remarks and some ideas on new directions to explore are given. In Appendix A are briefly reported some concepts and results about Discrete Event Systems which should help the reader in understanding some crucial points in chapter 5; while in Appendix B an overview on the experimental testbed of the Laboratory of Automation of University of Bologna, is reported to validated the approach presented in chapter 3, chapter 4 and chapter 5. In Appendix C some components model used in chapter 5 for formal verification are reported.