Stochastic models and dynamic measures for the characterization of bistable circuits

During the last few years, a great deal of interest has risen concerning the applications of stochastic methods to several biochemical and biological phenomena. Phenomena like gene expression, cellular memory, bet-hedging strategy in bacterial growth and many others, cannot be described by continuous stochastic models due to their intrinsic discreteness and randomness. In this thesis I have used the Chemical Master Equation (CME) technique to modelize some feedback cycles and analyzing their properties, including experimental data. In the first part of this work, the effect of stochastic stability is discussed on a toy model of the genetic switch that triggers the cellular division, which malfunctioning is known to be one of the hallmarks of cancer. The second system I have worked on is the so-called futile cycle, a closed cycle of two enzymatic reactions that adds and removes a chemical compound, called phosphate group, to a specific substrate. I have thus investigated how adding noise to the enzyme (that is usually in the order of few hundred molecules) modifies the probability of observing a specific number of phosphorylated substrate molecules, and confirmed theoretical predictions with numerical simulations. In the third part the results of the study of a chain of multiple phosphorylation-dephosphorylation cycles will be presented. We will discuss an approximation method for the exact solution in the bidimensional case and the relationship that this method has with the thermodynamic properties of the system, which is an open system far from equilibrium.In the last section the agreement between the theoretical prediction of the total protein quantity in a mouse cells population and the observed quantity will be shown, measured via fluorescence microscopy.

Design methodologies of microwawe integrated circuits for satellite telecommunications

The running innovation processes of the microwave transistor technologies, used in the implementation of microwave circuits, have to be supported by the study and development of proper design methodologies which, depending on the applications, will fully exploit the technology potentialities. After the choice of the technology to be used in the particular application, the circuit designer has few degrees of freedom when carrying out his design; in the most cases, due to the technological constrains, all the foundries develop and provide customized processes optimized for a specific performance such as power, low-noise, linearity, broadband etc. For these reasons circuit design is always a “compromise”, an investigation for the best solution to reach a trade off between the desired performances. This approach becomes crucial in the design of microwave systems to be used in satellite applications; the tight space constraints impose to reach the best performances under proper electrical and thermal de-rated conditions, respect to the maximum ratings provided by the used technology, in order to ensure adequate levels of reliability. In particular this work is about one of the most critical components in the front-end of a satellite antenna, the High Power Amplifier (HPA). The HPA is the main power dissipation source and so the element which mostly engrave on space, weight and cost of telecommunication apparatus; it is clear from the above reasons that design strategies addressing optimization of power density, efficiency and reliability are of major concern. Many transactions and publications demonstrate different methods for the design of power amplifiers, highlighting the availability to obtain very good levels of output power, efficiency and gain. Starting from existing knowledge, the target of the research activities summarized in this dissertation was to develop a design methodology capable optimize power amplifier performances complying all the constraints imposed by the space applications, tacking into account the thermal behaviour in the same manner of the power and the efficiency. After a reminder of the existing theories about the power amplifier design, in the first section of this work, the effectiveness of the methodology based on the accurate control of the dynamic Load Line and her shaping will be described, explaining all steps in the design of two different kinds of high power amplifiers. Considering the trade-off between the main performances and reliability issues as the target of the design activity, we will demonstrate that the expected results could be obtained working on the characteristics of the Load Line at the intrinsic terminals of the selected active device. The methodology proposed in this first part is based on the assumption that designer has the availability of an accurate electrical model of the device; the variety of publications about this argument demonstrates that it is so difficult to carry out a CAD model capable to taking into account all the non-ideal phenomena which occur when the amplifier operates at such high frequency and power levels. For that, especially for the emerging technology of Gallium Nitride (GaN), in the second section a new approach for power amplifier design will be described, basing on the experimental characterization of the intrinsic Load Line by means of a low frequency high power measurements bench. Thanks to the possibility to develop my Ph.D. in an academic spin-off, MEC – Microwave Electronics for Communications, the results of this activity has been applied to important research programs requested by space agencies, with the aim support the technological transfer from universities to industrial world and to promote a science-based entrepreneurship. For these reasons the proposed design methodology will be explained basing on many experimental results.

Development of synthetic molecular circuits for the control of cell systems

Synthetic biology is a young field of applicative research aiming to design and build up artificial biological devices, useful for human applications. How synthetic biology emerged in past years and how the development of the Registry of Standard Biological Parts aimed to introduce one practical starting solution to apply the basics of engineering to molecular biology is presented in chapter 1 in the thesis The same chapter recalls how biological parts can make up a genetic program, the molecular cloning tecnique useful for this purpose, and an overview of the mathematical modeling adopted to describe gene circuit behavior. Although the design of gene circuits has become feasible the increasing complexity of gene networks asks for a rational approach to design gene circuits. A bottom-up approach was proposed, suggesting that the behavior of a complicated system can be predicted from the features of its parts. The option to use modular parts in large-scale networks will be facilitated by a detailed and shared characterization of their functional properties. Such a prediction, requires well-characterized mathematical models of the parts and of how they behave when assembled together. In chapter 2, the feasibility of the bottom-up approach in the design of a synthetic program in Escherichia coli bacterial cells is described. The rational design of gene networks is however far from being established. The synthetic biology approach can used the mathematical formalism to identify biological information not assessable with experimental measurements. In this context, chapter 3 describes the design of a synthetic sensor for identifying molecules of interest inside eukaryotic cells. The Registry of Standard parts collects standard and modular biological parts. To spread the use of BioBricks the iGEM competition was started. The ICM Laboratory, where Francesca Ceroni completed her Ph.D, partecipated with teams of students and Chapter 4 summarizes the projects developed.

A core calculus for the analysis and implementation of biologically inspired languages

The application of Concurrency Theory to Systems Biology is in its earliest stage of progress. The metaphor of cells as computing systems by Regev and Shapiro opened the employment of concurrent languages for the modelling of biological systems. Their peculiar characteristics led to the design of many bio-inspired formalisms which achieve higher faithfulness and specificity. In this thesis we present pi@, an extremely simple and conservative extension of the pi-calculus representing a keystone in this respect, thanks to its expressiveness capabilities. The pi@ calculus is obtained by the addition of polyadic synchronisation and priority to the pi-calculus, in order to achieve compartment semantics and atomicity of complex operations respectively. In its direct application to biological modelling, the stochastic variant of the calculus, Spi@, is shown able to model consistently several phenomena such as formation of molecular complexes, hierarchical subdivision of the system into compartments, inter-compartment reactions, dynamic reorganisation of compartment structure consistent with volume variation. The pivotal role of pi@ is evidenced by its capability of encoding in a compositional way several bio-inspired formalisms, so that it represents the optimal core of a framework for the analysis and implementation of bio-inspired languages. In this respect, the encodings of BioAmbients, Brane Calculi and a variant of P Systems in pi@ are formalised. The conciseness of their translation in pi@ allows their indirect comparison by means of their encodings. Furthermore it provides a ready-to-run implementation of minimal effort whose correctness is granted by the correctness of the respective encoding functions. Further important results of general validity are stated on the expressive power of priority. Several impossibility results are described, which clearly state the superior expressiveness of prioritised languages and the problems arising in the attempt of providing their parallel implementation. To this aim, a new setting in distributed computing (the last man standing problem) is singled out and exploited to prove the impossibility of providing a purely parallel implementation of priority by means of point-to-point or broadcast communication.

Intrinsic uncoupling in the ATP synthase of Escherichia coli. Studies on WT and ε-truncated mutants

The H+/ATP ratio in the catalysis of ATP synthase has generally been considered a fixed parameter. However, Melandri and coworkers have recently shown that, in the ATP synthase of the photosynthetic bacterium Rb.capsulatus, this ratio can significantly decrease during ATP hydrolysis when the concentration of either ADP or Pi is maintained at a low level (Turina et al., 2004). The present work has dealt with the ATP synthase of E.coli, looking for evidence of this phenomenon of intrinsic uncoupling in this organism as well. First of all, we have shown that the DCCD-sensitive ATP hydrolysis activity of E.coli internal membranes was strongly inhibited by ADP and Pi, with a half-maximal effect in the submicromolar range for ADP and at 140 µM for Pi. In contrast to this monotonic inhibition, however, the proton pumping activity of the enzyme, as estimated under the same conditions by the fluorescence quenching of the ΔpH-sensitive probe ACMA, showed a clearly biphasic progression, both for Pi, increasing from 0 up to approximately 200 µM, and for ADP, increasing from 0 up to a few µM. We have interpreted these results as indicating that the occupancy of ADP and Pi binding sites shifts the enzyme from a partially uncoupled state to a fully coupled state, and we expect that the ADP- and Pi-modulated intrinsic uncoupling is likely to be a general feature of prokaryotic ATP synthases. Moreover, the biphasicity of the proton pumping data suggested that two Pi binding sites are involved. In order to verify whether the same behaviour could be observed in the isolated enzyme, we have purified the ATP synthase of E.coli and reconstituted it into liposomes. Similarly as observed in the internal membrane preparation, in the isolated and reconstituted enzyme it was possible to observe inhibition of the hydrolytic activity by ADP and Pi (with half-maximal effects at few µM for ADP and at 400 µM for Pi) with a concomitant stimulation of proton pumping. Both the inhibition of ATP hydrolysis and the stimulation of proton pumping as a function of Pi were lost upon ADP removal by an ADP trap. These data have made it possible to conclude that the results obtained in E.coli internal membranes are not due to the artefactual interference of enzymatic activities other than the ones of the ATP synthase. In addition, data obtained with liposomes have allowed a calibration of the ACMA signal by ΔpH transitions of known extent, leading to a quantitative evaluation of the proton pumping data. Finally, we have focused our efforts on searching for a possible structural candidate involved in the phenomenon of intrinsic uncoupling. The ε-subunit of the ATP-synthase is known as an endogenous inhibitor of the hydrolysis activity of the complex and appears to undergo drastic conformational changes between a non-inhibitory form (down-state) and an inhibitory form (up-state)(Rodgers & Wilce, 2000; Gibbons et al., 2000). In addition, the results of Cipriano & Dunn (2006) indicated that the C-terminal domain of this subunit played an important role in the coupling mechanism of the pump, and those of Capaldi et al. (2001), Suzuki et al. (2003) were consistent with the down-state showing a higher hydrolysis-to-synthesis ratio than the up-state. Therefore, we decided to search for modulation of pumping efficiency in a C-terminally truncated ε mutant. A low copy number expression vector has been built, carrying an extra copy of uncC, with the aim of generating an ε-overexpressing E.coli strain in which normal levels of assembly of the mutated ATP-synthase complex would be promoted. We have then compared the ATP hydrolysis and the proton pumping activity in membranes prepared from these ε-overexpressing E.coli strains, which carried either the WT ε subunit or the ε88-stop truncated form. Both strains yielded well energized membranes. Noticeably, they showed a marked difference in the inhibition of hydrolysis by Pi, this effect being largely lost in the truncated mutant. However, pre-incubation of the mutated enzyme with ADP at low nanomolar concentrations (apparent Kd = 0.7nM) restored the hydrolysis inhibition, together with the modulation of intrinsic uncoupling by Pi, indicating that, contrary to wild-type, during membrane preparation the truncated mutant had lost the ADP bound at this high-affinity site, evidently due to a lower affinity (and/or higher release) for ADP of the mutant relative to wild type. Therefore, one of the effects of the C-terminal domain of ε appears to be to modulate the affinity of at least one of the binding sites for ADP. The lack of this domain does not appear so much to influence the modulability of coupling efficiency, but instead the extent of this modulation. At higher preincubated ADP concentrations (apparent Kd = 117nM), the only observed effects were inhibition of both hydrolysis and synthesis, providing a direct proof that two ADP-binding sites on the enzyme are involved in the inhibition of hydrolysis, of which only the one at higher affinity also modulates the coupling efficiency.

Epidemiological and functional characterization of Streptococcus pneumoniae pili

Streptococcus pneumoniae is an important life threatening human pathogen causing agent of invasive diseases such as otitis media, pneumonia, sepsis and meningitis, but is also a common inhabitant of the respiratory tract of children and healthy adults. Likewise most streptococci, S. pneumoniae decorates its surface with adhesive pili, composed of covalently linked subunits and involved in the attachment to epithelial cells and virulence. The pneumococcal pili are encoded by two genomic regions, pilus islet 1 (PI-1), and pilus islet-2 (PI-2), which are present in about 30% and 16% of the pneumococcal strains, respectively. PI-1 exists in three clonally related variants, whereas PI-2 is highly conserved. The presence of the islets does not correlate with the serotype of the strains, but with the genotype (as determined by Multi Locus Sequence Typing). The prevalence of PI-1 and PI-2 positive strains is similar in isolates from invasive disease and carriage. To better dissect a possible association between PIs presence and disease we evaluated the distribution of the two PIs in a panel of 113 acute otitis media (AOM) clinical isolates from Israel. PI-1 was present in 30.1% (N=34) of the isolates tested, and PI-2 in 7% (N=8). We found that 50% of the PI-1 positive isolates belonged to the international clones Spain9V-3 (ST156) and Taiwan19F-14 (ST236), and that PI-2 was not present in the absence of Pl-1. In conclusion, there was no correlation between PIs presence and AOM, and, in general, the observed differences in PIs prevalence are strictly dependent upon regional differences in the distribution of the clones. Finally, in the AOM collection the prevalence of PI-1 was higher among antibiotic resistant isolates, confirming previous indications obtained by the in silico analysis of the MLST database collection. Since the pilus-1 subunits were shown to confer protection in mouse models of infection both in active and passive immunization studies, and were regarded as potential candidates for a new generation of protein-based vaccines, the functional characterization was mainly focused on S. pneumoniae pilus -1 components. The pneumococcal pilus-1 is composed of three subunits, RrgA, RrgB and RrgC, each stabilized by intra-molecular isopeptide bonds and covalently polymerized by means of inter-molecular isopeptide bonds to form an extended fibre. The pilus shaft is a multimeric structure mainly composed by the RrgB backbone subunit. The minor ancillary proteins are located at the tip and at the base of the pilus, where they have been proposed to act as the major adhesin (RrgA) and as the pilus anchor (RrgC), respectively. RrgA is protective in in vivo mouse models, and exists in two variants (clades I and II). Mapping of the sequence variability onto the RrgA structure predicted from X-ray data showed that the diversity was restricted to the “head” of the protein, which contains the putative binding domains, whereas the elongated “stalk” was mostly conserved. To investigate whether this variability could influence the adhesive capacity of RrgA and to map the regions important for binding, two full-length protein variants and three recombinant RrgA portions were tested for adhesion to lung epithelial cells and to purified extracellular matrix (ECM) components. The two RrgA variants displayed similar binding abilities, whereas none of the recombinant fragments adhered at levels comparable to those of the full-length protein, suggesting that proper folding and structural arrangement are crucial to retain protein functionality. Furthermore, the two RrgA variants were shown to be cross-reactive in vitro and cross-protective in vivo in a murine model of passive immunization. Taken together, these data indicate that the region implicated in adhesion and the functional epitopes responsible for the protective ability of RrgA may be conserved and that the considerable level of variation found within the “head” domain of RrgA may have been generated by immunologic pressure without impairing the functional integrity of the pilus.

Nano-Power Integrated Circuits for Energy Harvesting

The energy harvesting research field has grown considerably in the last decade due to increasing interests in energy autonomous sensing systems, which require smart and efficient interfaces for extracting power from energy source and power management (PM) circuits. This thesis investigates the design trade-offs for minimizing the intrinsic power of PM circuits, in order to allow operation with very weak energy sources. For validation purposes, three different integrated power converter and PM circuits for energy harvesting applications are presented. They have been designed for nano-power operations and single-source converters can operate with input power lower than 1 μW. The first IC is a buck-boost converter for piezoelectric transducers (PZ) implementing Synchronous Electrical Charge Extraction (SECE), a non-linear energy extraction technique. Moreover, Residual Charge Inversion technique is exploited for extracting energy from PZ with weak and irregular excitations (i.e. lower voltage), and the implemented PM policy, named Two-Way Energy Storage, considerably reduces the start-up time of the converter, improving the overall conversion efficiency. The second proposed IC is a general-purpose buck-boost converter for low-voltage DC energy sources, up to 2.5 V. An ultra-low-power MPPT circuit has been designed in order to track variations of source power. Furthermore, a capacitive boost circuit has been included, allowing the converter start-up from a source voltage VDC0 = 223 mV. A nano-power programmable linear regulator is also included in order to provide a stable voltage to the load. The third IC implements an heterogeneous multisource buck-boost converter. It provides up to 9 independent input channels, of which 5 are specific for PZ (with SECE) and 4 for DC energy sources with MPPT. The inductor is shared among channels and an arbiter, designed with asynchronous logic to reduce the energy consumption, avoids simultaneous access to the buck-boost core, with a dynamic schedule based on source priority.

Synthesis and conformational analysis of heteroaromatic atropisomeric systems

The research work reported in this Thesis was held along two main lines of research. The first and main line of research is about the synthesis of heteroaromatic compounds with increasing steric hindrance, with the aim of preparing stable atropisomers. The main tools used for the study of these dynamic systems, as described in the Introduction, are DNMR, coupled with line shape simulation and DFT calculations, aimed to the conformational analysis for the prediction of the geometries and energy barriers to the trasition states. This techniques have been applied to the research projects about: • atropisomers of arylmaleimides; • atropisomers of 4-arylpyrazolo[3,4-b]pyridines; • study of the intramolecular NO2/CO interaction in solution; • study on 2-arylpyridines. Parallel to the main project, in collaboration with other groups, the research line about determination of the absolute configuration was followed. The products, deriving form organocatalytic reactions, in many cases couldn’t be analyzed by means of X-Ray diffraction, making necessary the development of a protocol based on spectroscopic methodologies: NMR, circular dichroism and computational tools (DFT, TD-DFT) have been implemented in this scope. In this Thesis are reported the determination of the absolute configuration of: • substituted 1,2,3,4-tetrahydroquinolines; • compounds from enantioselective Friedel-Crafts alkylation-acetalization cascade of naphthols with α,β-unsaturated cyclic ketones; • substituted 3,4-annulated indoles.

The impact of phosphoinositide metabolic enzymes in glioblastoma: a tale of phosphoinositide-specific phospholipase C PLCβ1 and 5-phosphatase SKIP

Background: Glioblastoma multiforme (GBM) is one of the deadliest and most aggressive form of primary brain tumor. Unfortunately, current GBM treatment therapies are not effective in treating GBM patients. They usually experience very poor prognosis with a median survival of approximately 12 months. Only 3-5% survive up to 3 years or more. A large-scale gene profile study revealed that several genes involved in essential cellular processes are altered in GBM, thus, explaining why existing therapies are not effective. The survival of GBM patients depends on understanding the molecular and key signaling events associated with these altered physiological processes in GBM. Phosphoinositides (PI) form just a tiny fraction of the total lipid content in humans, however they are implicated in almost all essential biological processes, such as acting as second messengers in spatio-temporal regulation of cell signaling, cytoskeletal reorganization, cell adhesion, migration, apoptosis, vesicular trafficking, differentiation, cell cycle and post-translational modifications. Interestingly, these essential processes are altered in GBM. More importantly, incoming reports have associated PI metabolism, which is mediated by several PI phosphatases such as SKIP, lipases such as PLCβ1, and other kinases, to regulate GBM associated cellular processes. Even as PLCβ1 and SKIP are involved in regulating aberrant cellular processes in several other cancers, very few studies, of which majority are in-silico-based, have focused on the impact of PLCβ1 and SKIP in GBM. Hence, it is important to employ clinical, in vitro, and in vivo GBM models to define the actual impact of PLCβ1 and SKIP in GBM. AIM: Since studies of PLCβ1 and SKIP in GBM are limited, this study aimed at determining the pathological impact of PI metabolic enzymes, PLCB1 and SKIP, in GBM patient samples, GBM cell line models, and xenograft models for SKIP. Results: For the first time, this study confirmed through qPCR that PLCβ1 gene expression is lower in human GBM patient samples. Moreover, PLCβ1 gene expression inversely correlates with pathological grades of glioma; it decreases as glioma grades increases or worsens. Silencing PLCβ1 in U87MG GBM cells produces a dual impact in GBM by participating in both pro-tumoral and anti-tumoral roles. PLCβ1 knockdown cells were observed to have more migratory abilities, increased cell to extracellular matrix (ECM) adhesion, transition from epithelial phenotype to mesenchymal phenotype through the upregulation of EMT transcription factors Twist1 and Slug, and mesenchymal marker, vimentin. On the other hand, p-Akt and p-mTOR protein expression were downregulated in PLCβ1 knockdown cells. Thus, the oncogenic pathway PI3K/Akt/mTOR pathway is inhibited during PLCβ1 knockdown. Consistently, cell viability in PLCβ1 knockdown cells were significantly decreased compared to controls. As for SKIP, this study demonstrated that about 48% of SKIP colocalizes with nuclear PtdIns(4,5)P2 to nuclear speckles and that SKIP knockdown alters nuclear PtdIns(4,5)P2 in a cell-type dependent manner. In addition, SKIP silencing increased tumor volume and weight in xenografts than controls by reducing apoptosis and increasing viability. All in all, these data confirm that PLCβ1 and SKIP are involved in GBM pathology and a complete understanding of their roles in GBM may be beneficial.

A preclinical study of spinal cord injury focused on cellular and molecular modifications as potential targets for innovative therapies

Spinal Cord Injury (SCI) is a devastating condition for human and animal health. In SCI particularly, neurons, oligodendrocytes precursor cells, and mature oligodendrocytes are highly vulnerable to the toxic microenvironment after the lesion and susceptible to the elevated levels of noxious stimuli. Thus the regenerative response of the organism in case of SCI is significantly reduced, and only little spontaneous amelioration is observed in lesioned patients during the early phases. This work mainly focuses on studying and characterizing the modification induced by the SCI in a preclinical animal model. We investigated the ECM composition in the spinal cord segments surrounding the primary lesion site at a gene expression level. We found Timp1 and CD44 as a crucial hub in the secondary cascade of SCI in both spinal cord segments surrounding the lesion site. Interestingly, a temporal and anatomical difference in gene expression, indicating a complex regulation of ECM genes after SCI that could be used as a tool for regenerative medicine. We also investigated the modification in synaptic plasticity-related gene expression in spinal and supraspinal areas involved in motor control. We confirmed the anatomical and temporal difference in gene expression in spinal cord tissue. This analysis suggests that a molecular mapping of the lesion-induced modification could be a useful tool for regenerative medicine. In the last part, we evaluated the efficacy of an implantable biopolymer loaded with an anti-inflammatory drug and a pro-myelinating agent on the acute phase of SCI in our preclinical model. We found a consistent reduction of the inflammatory state in the spinal lesion site and the cord's surrounding segments. Moreover, we found increased preservation of the spinal cord tissue with a related upregulation of neuronal and oligodendroglial markers after lesion. Our treatment showed effective ameliorating functional outcome and reducing the lesion extension in the chronic phase.

About stabilization of non-minimum phase systems by output feedback

This thesis work has been motivated by an internal benchmark dealing with the output regulation problem of a nonlinear non-minimum phase system in the case of full-state feedback. The system under consideration structurally suffers from finite escape time, and this condition makes the output regulation problem very hard even for very simple steady-state evolution or exosystem dynamics, such as a simple integrator. This situation leads to studying the approaches developed for controlling Non-minimum phase systems and how they affect feedback performances. Despite a lot of frequency domain results, only a few works have been proposed for describing the performance limitations in a state space system representation. In particular, in our opinion, the most relevant research thread exploits the so-called Inner-Outer Decomposition. Such decomposition allows splitting the Non-minimum phase system under consideration into a cascade of two subsystems: a minimum phase system (the outer) that contains all poles of the original system and an all-pass Non-minimum phase system (the inner) that contains all the unavoidable pathologies of the unstable zero dynamics. Such a cascade decomposition was inspiring to start working on functional observers for linear and nonlinear systems. In particular, the idea of a functional observer is to exploit only the measured signals from the system to asymptotically reconstruct a certain function of the system states, without necessarily reconstructing the whole state vector. The feature of asymptotically reconstructing a certain state functional plays an important role in the design of a feedback controller able to stabilize the Non-minimum phase system.

Identificazione delle Lamine di tipo A come nuovi substrati della protein chinasi Akt/PKB

Akt (also called PKB) is a 63 kDa serine/threonine kinase involved in promotion of cell survival, proliferation a nd metabolic responses downstream the phosphoinositide-3-kinase (PI 3-kinase) signaling pathway. In resting cells, Akt is a predominantly cytosolic enzyme; however generation of PI 3-kinase lipid products recruits Akt to the plasma membrane, resulting in a conformational change which confers full enzymatic activity through the phosphorylation of the membrane-bound protein at two residues, Thr308, and Ser473. Activated Akt redistributes to cytoplasm and nucleus, where phosphorylation of specific substrates occurs. Both the presence and the activity of Akt in the nucleus have been described. An interesting mechanism that mediates nuclear translocation of Akt has been described in human mature T-cell leukemia: the product of TCL1 gene, Tcl1, interacts with the PH domain of phosphorylated Akt, thus driving Akt to the nucleus. In this context, Tcl1 may act as a direct transporter of Akt or may contribute to the formation of a complex that promotes the transport of active Akt to the nucleus, where it can phosphorylate nuclear substrates. A well described nuclear substrate if Foxo. IGF-1 triggers phosphorylation of Foxo by Akt inside the nucleus, where phospho-Foxo associates to 14.3.3 proteins that, in turn, promote its export to the cytoplasm where it is sequestered. Remarkably, Foxo phosphorylation by Akt has been shown to be a crucial event in Akt-dependent myogenesis. However, most Akt nuclear substrates have so far remained elusive, as well as nuclear Akt functions. This lack of information prompted us to undertake a search of substrates of Akt in the nucleus, by the combined use of 2D-separation/mass spectrometry and anti-Akt-phosphosubstrate antibody. This study presents evidence of A-type lamins as novel nuclear substrates of Akt. Lamins are type V intermediate filaments proteins found in the nucleus of higher eukaryotes where, together with lamin-binding proteins, they form the lamina at the nuclear envelope, providing mechanical stability for the nuclear membrane. By coimmunoprecipitation, it is demonstrated here that endogenous lamin A and Akt interact, and that A-type lamins are phosphorylated by Akt both in vitro and in vivo. Moreover, by phosphoaminoacid analysis and mutagenesis, it is further demonstrated that Akt phosphorylates lamin A at Ser404, and, more importantly, that while lamin A/C phosphorylation is stable throughout the cell cycle, phosphorylation of the precursor prelamin A becomes detectable as cells enter the G2 phase, picking at G2/M. This study also shows that lamin phosphorylation by Akt creates a binding site for 14.3.3 adaptors which, in turn, promote prelamin A degradation. While this mechanism is in agreement with a general role of Akt in the regulation of a subset of its substrates, opposite to what has been described, degradation is not mediated through a ubiquitination and proteasomal mechanism but through a lysosomal pathway, as indicated by the reverting action of the lysosomal inhibitor cloroquine. Phosphorylation is a key event in the mitotic breakdown of the nuclear lamina. However, the kinases and the precise sites of phosphorylation are scarcely known. Therefore, these results represent an important breakthrough in this very significant but understudied area. The phosphorylation of the precursor protein prelamin A and its subsequent degradation at G2/M, when both the nuclear envelop and the nuclear lamina disassemble, can be view as part of a mechanism to dispose off the precursor that is not needed in this precise context. The recently reported finding that patients affected by Emery-Dreifuss muscular dystrophy carry a mutation at Arg 401, in the Akt phosphorylation motif, open new perspective that warrant further investigation in this very important field.

Tecniche di progettazione tollerante alle variazioni per circuiti digitali in tecnologie nanometriche

The digital electronic market development is founded on the continuous reduction of the transistors size, to reduce area, power, cost and increase the computational performance of integrated circuits. This trend, known as technology scaling, is approaching the nanometer size. The lithographic process in the manufacturing stage is increasing its uncertainty with the scaling down of the transistors size, resulting in a larger parameter variation in future technology generations. Furthermore, the exponential relationship between the leakage current and the threshold voltage, is limiting the threshold and supply voltages scaling, increasing the power density and creating local thermal issues, such as hot spots, thermal runaway and thermal cycles. In addiction, the introduction of new materials and the smaller devices dimension are reducing transistors robustness, that combined with high temperature and frequently thermal cycles, are speeding up wear out processes. Those effects are no longer addressable only at the process level. Consequently the deep sub-micron devices will require solutions which will imply several design levels, as system and logic, and new approaches called Design For Manufacturability (DFM) and Design For Reliability. The purpose of the above approaches is to bring in the early design stages the awareness of the device reliability and manufacturability, in order to introduce logic and system able to cope with the yield and reliability loss. The ITRS roadmap suggests the following research steps to integrate the design for manufacturability and reliability in the standard CAD automated design flow: i) The implementation of new analysis algorithms able to predict the system thermal behavior with the impact to the power and speed performances. ii) High level wear out models able to predict the mean time to failure of the system (MTTF). iii) Statistical performance analysis able to predict the impact of the process variation, both random and systematic. The new analysis tools have to be developed beside new logic and system strategies to cope with the future challenges, as for instance: i) Thermal management strategy that increase the reliability and life time of the devices acting to some tunable parameter,such as supply voltage or body bias. ii) Error detection logic able to interact with compensation techniques as Adaptive Supply Voltage ASV, Adaptive Body Bias ABB and error recovering, in order to increase yield and reliability. iii) architectures that are fundamentally resistant to variability, including locally asynchronous designs, redundancy, and error correcting signal encodings (ECC). The literature already features works addressing the prediction of the MTTF, papers focusing on thermal management in the general purpose chip, and publications on statistical performance analysis. In my Phd research activity, I investigated the need for thermal management in future embedded low-power Network On Chip (NoC) devices.I developed a thermal analysis library, that has been integrated in a NoC cycle accurate simulator and in a FPGA based NoC simulator. The results have shown that an accurate layout distribution can avoid the onset of hot-spot in a NoC chip. Furthermore the application of thermal management can reduce temperature and number of thermal cycles, increasing the systemreliability. Therefore the thesis advocates the need to integrate a thermal analysis in the first design stages for embedded NoC design. Later on, I focused my research in the development of statistical process variation analysis tool that is able to address both random and systematic variations. The tool was used to analyze the impact of self-timed asynchronous logic stages in an embedded microprocessor. As results we confirmed the capability of self-timed logic to increase the manufacturability and reliability. Furthermore we used the tool to investigate the suitability of low-swing techniques in the NoC system communication under process variations. In this case We discovered the superior robustness to systematic process variation of low-swing links, which shows a good response to compensation technique as ASV and ABB. Hence low-swing is a good alternative to the standard CMOS communication for power, speed, reliability and manufacturability. In summary my work proves the advantage of integrating a statistical process variation analysis tool in the first stages of the design flow.

Effetti clinici e biologici della terapia epigenetica nelle sindromi mielodisplastiche a basso rischio

Background: Nucleoside 5-Azacitidine (5-Aza) in high risk MDS patients (pts) at a dose of 75mg/mq/day subcutaneously for 7 days, every 28 days, induces high hematologic response rates (hematologic improvement (HI) 50-60%, complete remission (CR) 10-30%) and prolongation of survival (at 2 years 50,8%). Aim: The role of 5-Aza in low-risk MDS patients is not well defined but its use in the earlier phases of disease could be more effective and useful to control the expansion of MDS clone and disease progression. In our phase II, prospective, multicentric trial a low-dose schedule of 5-Aza (75 mg/mq daily for 5 consecutive days every 28 days) was given to low-risk MDS pts in order to evaluate its efficacy and tolerability and to identify biological markers to predict the response. Methods: From September 2008 to February 2010, 34 patients were enrolled into the study. Fifteen patients had refractory anemia (RA), 5 patients refractory anemia with ringed sideroblasts (RARS), 7 patients refractory cytopenia with multilineage dysplasia (RCMD) and 7 patients refractory anemia with excess blasts-1 (RAEB-1). All patients failed previously EPO therapy and were in chronic red blood cell (RBC) supportive care with a median transfusions requirement of 4 units/monthly. The response treatment criteria was according to IWG 2006. Results: At present time 31 out of 34 pts are evaluable: 12/31 pts (39%) completed the treatment plan (8 courses), 7/31 pts (22%) performed the first 4 courses, 8/31 (26%) made 1 to 3 courses and 4/31 (13%) died during the treatment period. Out of 12 pts who completed the 8 courses of therapy 10 (83%) obtained an HI, 2/12 (17%) maintained a stable disease. Out of 10 pts who obtained HI, 4 pts (40%) achieved a CR. Generally the drug was very well tolerated. The most commonly reported hematologic toxicities were neutropenia (55%) and thrombocytopenia (19%) but they were transitory and usually no delay of treatment was necessary. 2/4 pts died early after the 1th cycle for septic shock and gastrointestinal hemorrage respectively whereas 2/4 pts died in a condition of stable disease after the 4th cycle for pneumonia and respiratory distress. Samples for biologic studies have been collected from the pts before starting the therapy and at the end of 4th and 8th course. Preliminary data on the lipid signalling pathways suggested a direct correlation between PI-PLC-β1 gene expression and 5-Aza responsiveness. Conclusion: Interim analysis of our study based on the small number of cases who completed the treatment program, shows that 83% of pts obtain an HI and 40% obtain a CR. 4 patients died during the treatment and even if the causes were reported as no related to the therapy it has been considered that caution has to be reserved in given 5-Aza in these pts who are elderly and frail. Preliminary data of PI-PLC-β1 gene expression suggest that this and probably other biological markers could help us to know a priori who are the patients who have more chances to respond.

Studio di espressione e funzione della PLCβ1 nucleare in modelli murini ed umani di cellule ematopoietiche mieloidi e linfoidi

The aims of this work were to investigate the role of nuclear Phospholipase C beta 1 (PI-PLCβ1) in human and mouse cell lines and to identify new binding partners of nuclear PI-PLCβ1 to further understand the functional network in which the enzyme acts. The intracellular distribution of PI-PLCβ1 was further investigated in human leukaemia cell lines (NB4, HL60, THP1, CEM, Jurkat, K562). With the exception of HL60, a high endogenous level of PI-PLCβ1 was detected in purified nuclei in each of the cell lines. We found that also in Ba/F3 pro-B cells overexpressing PI-PLCβ1b the protein localize within the nucleus. Although our data demonstrated that PI-PLCβ1b was not involved in cell proliferation and IGF-1 response as shown in other cell lines (FELC and Swiss 3T3), there was an effect on apoptosis. Activation of early apoptotic markers caspase-3 and PARP was delayed in PI-PLCβ1b overexpressing Ba/F3 cells treated with 5 gr/ml mitomycin C for 24h. We performed an antibody-specific immunoprecipitation on nuclear lysates from FELC-PLCβ1b cells. Mass spectrometry analysis (nano-ESI-Q-TOF) of co-immunoprecipitated proteins allowed for identification of 92 potential nuclear PI-PLCβ1b interactors. Among these, several already documented PI-PLCβ1b interacting partners (Srp20, LaminB, EF1α2) were identified, further validating our data. All the identified proteins were nuclear, mostly localized within the nuclear speckles. This evidence is particularly relevant as PI-PLCβ1 is known to localize in the same domains. Many of the identified proteins are involved in cell cycle, proliferation and transcriptional control. In particular, many of the proteins are components of the spliceosome multi-complex, strengthening the idea that PI-PLCβ1b is involved in mRNA processing and maturation. Future work will aim to better characterize the regulatory role of PI-PLCβ1b in mRNA splicing.