RNA Interference and cyclooxygenase-2 (COX-2) regulation in colon cancer cells

Despite new methods and combined strategies, conventional cancer chemotherapy still lacks specificity and induces drug resistance. Gene therapy can offer the potential to obtain the success in the clinical treatment of cancer and this can be achieved by replacing mutated tumour suppressor genes, inhibiting gene transcription, introducing new genes encoding for therapeutic products, or specifically silencing any given target gene. Concerning gene silencing, attention has recently shifted onto the RNA interference (RNAi) phenomenon. Gene silencing mediated by RNAi machinery is based on short RNA molecules, small interfering RNAs (siRNAs) and microRNAs (miRNAs), that are fully o partially homologous to the mRNA of the genes being silenced, respectively. On one hand, synthetic siRNAs appear as an important research tool to understand the function of a gene and the prospect of using siRNAs as potent and specific inhibitors of any target gene provides a new therapeutical approach for many untreatable diseases, particularly cancer. On the other hand, the discovery of the gene regulatory pathways mediated by miRNAs, offered to the research community new important perspectives for the comprehension of the physiological and, above all, the pathological mechanisms underlying the gene regulation. Indeed, changes in miRNAs expression have been identified in several types of neoplasia and it has also been proposed that the overexpression of genes in cancer cells may be due to the disruption of a control network in which relevant miRNA are implicated. For these reasons, I focused my research on a possible link between RNAi and the enzyme cyclooxygenase-2 (COX-2) in the field of colorectal cancer (CRC), since it has been established that the transition adenoma-adenocarcinoma and the progression of CRC depend on aberrant constitutive expression of COX-2 gene. In fact, overexpressed COX-2 is involved in the block of apoptosis, the stimulation of tumor-angiogenesis and promotes cell invasion, tumour growth and metastatization. On the basis of data reported in the literature, the first aim of my research was to develop an innovative and effective tool, based on the RNAi mechanism, able to silence strongly and specifically COX-2 expression in human colorectal cancer cell lines. In this study, I firstly show that an siRNA sequence directed against COX-2 mRNA (siCOX-2), potently downregulated COX-2 gene expression in human umbilical vein endothelial cells (HUVEC) and inhibited PMA-induced angiogenesis in vitro in a specific, non-toxic manner. Moreover, I found that the insertion of a specific cassette carrying anti-COX-2 shRNA sequence (shCOX-2, the precursor of siCOX-2 previously tested) into a viral vector (pSUPER.retro) greatly increased silencing potency in a colon cancer cell line (HT-29) without activating any interferon response. Phenotypically, COX-2 deficient HT-29 cells showed a significant impairment of their in vitro malignant behaviour. Thus, results reported here indicate an easy-to-use, powerful and high selective virus-based method to knockdown COX-2 gene in a stable and long-lasting manner, in colon cancer cells. Furthermore, they open up the possibility of an in vivo application of this anti-COX-2 retroviral vector, as therapeutic agent for human cancers overexpressing COX-2. In order to improve the tumour selectivity, pSUPER.retro vector was modified for the shCOX-2 expression cassette. The aim was to obtain a strong, specific transcription of shCOX-2 followed by COX-2 silencing mediated by siCOX-2 only in cancer cells. For this reason, H1 promoter in basic pSUPER.retro vector [pS(H1)] was substituted with the human Cox-2 promoter [pS(COX2)] and with a promoter containing repeated copies of the TCF binding element (TBE) [pS(TBE)]. These promoters were choosen because they are partculary activated in colon cancer cells. COX-2 was effectively silenced in HT-29 and HCA-7 colon cancer cells by using enhanced pS(COX2) and pS(TBE) vectors. In particular, an higher siCOX-2 production followed by a stronger inhibition of Cox-2 gene were achieved by using pS(TBE) vector, that represents not only the most effective, but also the most specific system to downregulate COX-2 in colon cancer cells. Because of the many limits that a retroviral therapy could have in a possible in vivo treatment of CRC, the next goal was to render the enhanced RNAi-mediate COX-2 silencing more suitable for this kind of application. Xiang and et al. (2006) demonstrated that it is possible to induce RNAi in mammalian cells after infection with engineered E. Coli strains expressing Inv and HlyA genes, which encode for two bacterial factors needed for successful transfer of shRNA in mammalian cells. This system, called “trans-kingdom” RNAi (tkRNAi) could represent an optimal approach for the treatment of colorectal cancer, since E. Coli in normally resident in human intestinal flora and could easily vehicled to the tumor tissue. For this reason, I tested the improved COX-2 silencing mediated by pS(COX2) and pS(TBE) vectors by using tkRNAi system. Results obtained in HT-29 and HCA-7 cell lines were in high agreement with data previously collected after the transfection of pS(COX2) and pS(TBE) vectors in the same cell lines. These findings suggest that tkRNAi system for COX-2 silencing, in particular mediated by pS(TBE) vector, could represent a promising tool for the treatment of colorectal cancer. Flanking the studies addressed to the setting-up of a RNAi-mediated therapeutical strategy, I proposed to get ahead with the comprehension of new molecular basis of human colorectal cancer. In particular, it is known that components of the miRNA/RNAi pathway may be altered during the progressive development of colorectal cancer (CRC), and it has been already demonstrated that some miRNAs work as tumor suppressors or oncomiRs in colon cancer. Thus, my hypothesis was that overexpressed COX-2 protein in colon cancer could be the result of decreased levels of one or more tumor suppressor miRNAs. In this thesis, I clearly show an inverse correlation between COX-2 expression and the human miR- 101(1) levels in colon cancer cell lines, tissues and metastases. I also demonstrate that the in vitro modulating of miR-101(1) expression in colon cancer cell lines leads to significant variations in COX-2 expression, and this phenomenon is based on a direct interaction between miR-101(1) and COX-2 mRNA. Moreover, I started to investigate miR-101(1) regulation in the hypoxic environment since adaptation to hypoxia is critical for tumor cell growth and survival and it is known that COX-2 can be induced directly by hypoxia-inducible factor 1 (HIF-1). Surprisingly, I observed that COX-2 overexpression induced by hypoxia is always coupled to a significant decrease of miR-101(1) levels in colon cancer cell lines, suggesting that miR-101(1) regulation could be involved in the adaption of cancer cells to the hypoxic environment that strongly characterize CRC tissues.

Code synchronization and interference management techniques for satellite navigation and communications

This thesis collects the outcomes of a Ph.D. course in Telecommunications engineering and it is focused on enabling techniques for Spread Spectrum (SS) navigation and communication satellite systems. It provides innovations for both interference management and code synchronization techniques. These two aspects are critical for modern navigation and communication systems and constitute the common denominator of the work. The thesis is organized in two parts: the former deals with interference management. We have proposed a novel technique for the enhancement of the sensitivity level of an advanced interference detection and localization system operating in the Global Navigation Satellite System (GNSS) bands, which allows the identification of interfering signals received with power even lower than the GNSS signals. Moreover, we have introduced an effective cancellation technique for signals transmitted by jammers, exploiting their repetitive characteristics, which strongly reduces the interference level at the receiver. The second part, deals with code synchronization. More in detail, we have designed the code synchronization circuit for a Telemetry, Tracking and Control system operating during the Launch and Early Orbit Phase; the proposed solution allows to cope with the very large frequency uncertainty and dynamics characterizing this scenario, and performs the estimation of the code epoch, of the carrier frequency and of the carrier frequency variation rate. Furthermore, considering a generic pair of circuits performing code acquisition, we have proposed a comprehensive framework for the design and the analysis of the optimal cooperation procedure, which minimizes the time required to accomplish synchronization. The study results particularly interesting since it enables the reduction of the code acquisition time without increasing the computational complexity. Finally, considering a network of collaborating navigation receivers, we have proposed an innovative cooperative code acquisition scheme, which allows exploit the shared code epoch information between neighbor nodes, according to the Peer-to-Peer paradigm.

The APSEL4D Monolithic Active Pixel Sensor and its Usage in a Single Electron Interference Experiment

We have realized a Data Acquisition chain for the use and characterization of APSEL4D, a 32 x 128 Monolithic Active Pixel Sensor, developed as a prototype for frontier experiments in high energy particle physics. In particular a transition board was realized for the conversion between the chip and the FPGA voltage levels and for the signal quality enhancing. A Xilinx Spartan-3 FPGA was used for real time data processing, for the chip control and the communication with a Personal Computer through a 2.0 USB port. For this purpose a firmware code, developed in VHDL language, was written. Finally a Graphical User Interface for the online system monitoring, hit display and chip control, based on windows and widgets, was realized developing a C++ code and using Qt and Qwt dedicated libraries. APSEL4D and the full acquisition chain were characterized for the first time with the electron beam of the transmission electron microscope and with 55Fe and 90Sr radioactive sources. In addition, a beam test was performed at the T9 station of the CERN PS, where hadrons of momentum of 12 GeV/c are available. The very high time resolution of APSEL4D (up to 2.5 Mfps, but used at 6 kfps) was fundamental in realizing a single electron Young experiment using nanometric double slits obtained by a FIB technique. On high statistical samples, it was possible to observe the interference and diffractions of single isolated electrons traveling inside a transmission electron microscope. For the first time, the information on the distribution of the arrival time of the single electrons has been extracted.

Interference Management and Energy Efficiency in Satellite Communications

The main areas of research of this thesis are Interference Management and Link-Level Power Efficiency for Satellite Communications. The thesis is divided in two parts. Part I tackles the problem of interference environments in satellite communications, and interference mitigation strategies, not just in terms of avoidance of the interferers, but also in terms of actually exploiting the interference present in the system as a useful signal. The analysis follows a top-down approach across different levels of investigation, starting from system level consideration on interference management, down to link-level aspects and to intra-receiver design. Interference Management techniques are proposed at all the levels of investigation, with interesting results. Part II is related to efficiency in the power domain, for instance in terms of required Input Back-off at the power amplifiers, which can be an issue for waveform based on linear modulations, due to their varying envelope. To cope with such aspects, an analysis is carried out to compare linear modulation with waveforms based on constant envelope modulations. It is shown that in some scenarios, constant envelope waveforms, even if at lower spectral efficiency, outperform linear modulation waveform in terms of energy efficiency.

GNSS interference management techniques against malicious attacks

This thesis collects the outcomes of a Ph.D. course in Telecommunications Engineering and it is focused on the study and design of possible techniques able to counteract interference signal in Global Navigation Satellite System (GNSS) systems. The subject is the jamming threat in navigation systems, that has become a very increasingly important topic in recent years, due to the wide diffusion of GNSS-based civil applications. Detection and mitigation techniques are developed in order to fight out jamming signals, tested in different scenarios and including sophisticated signals. The thesis is organized in two main parts, which deal with management of GNSS intentional counterfeit signals. The first part deals with the interference management, focusing on the intentional interfering signal. In particular, a technique for the detection and localization of the interfering signal level in the GNSS bands in frequency domain has been proposed. In addition, an effective mitigation technique which exploits the periodic characteristics of the common jamming signals reducing interfering effects at the receiver side has been introduced. Moreover, this technique has been also tested in a different and more complicated scenario resulting still effective in mitigation and cancellation of the interfering signal, without high complexity. The second part still deals with the problem of interference management, but regarding with more sophisticated signal. The attention is focused on the detection of spoofing signal, which is the most complex among the jamming signal types. Due to this highly difficulty in detect and mitigate this kind of signal, spoofing threat is considered the most dangerous. In this work, a possible techniques able to detect this sophisticated signal has been proposed, observing and exploiting jointly the outputs of several operational block measurements of the GNSS receiver operating chain.

NLGA based Active Control in Aerospace: fault tolerability, disturbance rejection, and parameter estimation

A new control scheme has been presented in this thesis. Based on the NonLinear Geometric Approach, the proposed Active Control System represents a new way to see the reconfigurable controllers for aerospace applications. The presence of the Diagnosis module (providing the estimation of generic signals which, based on the case, can be faults, disturbances or system parameters), mean feature of the depicted Active Control System, is a characteristic shared by three well known control systems: the Active Fault Tolerant Controls, the Indirect Adaptive Controls and the Active Disturbance Rejection Controls. The standard NonLinear Geometric Approach (NLGA) has been accurately investigated and than improved to extend its applicability to more complex models. The standard NLGA procedure has been modified to take account of feasible and estimable sets of unknown signals. Furthermore the application of the Singular Perturbations approximation has led to the solution of Detection and Isolation problems in scenarios too complex to be solved by the standard NLGA. Also the estimation process has been improved, where multiple redundant measuremtent are available, by the introduction of a new algorithm, here called "Least Squares - Sliding Mode". It guarantees optimality, in the sense of the least squares, and finite estimation time, in the sense of the sliding mode. The Active Control System concept has been formalized in two controller: a nonlinear backstepping controller and a nonlinear composite controller. Particularly interesting is the integration, in the controller design, of the estimations coming from the Diagnosis module. Stability proofs are provided for both the control schemes. Finally, different applications in aerospace have been provided to show the applicability and the effectiveness of the proposed NLGA-based Active Control System.

Distraction by task-irrelevant stimuli: the effects of endogenous spatial attention and visual working memory load

Salient stimuli, like sudden changes in the environment or emotional stimuli, generate a priority signal that captures attention even if they are task-irrelevant. However, to achieve goal-driven behavior, we need to ignore them and to avoid being distracted. It is generally agreed that top-down factors can help us to filter out distractors. A fundamental question is how and at which stage of processing the rejection of distractors is achieved. Two circumstances under which the allocation of attention to distractors is supposed to be prevented are represented by the case in which distractors occur at an unattended location (as determined by the deployment of endogenous spatial attention) and when the amount of visual working memory resources is reduced by an ongoing task. The present thesis is focused on the impact of these factors on three sources of distraction, namely auditory and visual onsets (Experiments 1 and 2, respectively) and pleasant scenes (Experiment 3). In the first two studies we recorded neural correlates of distractor processing (i.e., Event-Related Potentials), whereas in the last study we used interference effects on behavior (i.e., a slowing down of response times on a simultaneous task) to index distraction. Endogenous spatial attention reduced distraction by auditory stimuli and eliminated distraction by visual onsets. Differently, visual working memory load only affected the processing of visual onsets. Emotional interference persisted even when scenes occurred always at unattended locations and when visual working memory was loaded. Altogether, these findings indicate that the ability to detect the location of salient task-irrelevant sounds and identify the affective significance of natural scenes is preserved even when the amount of visual working memory resources is reduced by an ongoing task and when endogenous attention is elsewhere directed. However, these results also indicate that the processing of auditory and visual distractors is not entirely automatic.

Interference mitigation techniques for cloud-RAN deployments in 5G and beyond systems

The deployment of ultra-dense networks is one of the most promising solutions to manage the phenomenon of co-channel interference that affects the latest wireless communication systems, especially in hotspots. To meet the requirements of the use-cases and the immense amount of traffic generated in these scenarios, 5G ultra-dense networks are being deployed using various technologies, such as distributed antenna system (DAS) and cloud-radio access network (C-RAN). Through these centralized densification schemes, virtualized baseband processing units coordinate the distributed access points and manage the available network resources. In particular, link adaptation techniques are shown to be fundamental to overall system operation and performance enhancement. The core of this dissertation is the result of an analysis and a comparison of dynamic and adaptive methods for modulation and coding scheme (MCS) selection applied to the latest mobile telecommunications standards. A novel algorithm based on the proportional-integral-derivative (PID) controller principles and block error rate (BLER) target has been proposed. Tests were conducted in a 4G and 5G system level laboratory and, by means of a channel emulator, the performance was evaluated for different channel models and target BLERs. Furthermore, due to the intrinsic sectorization of the end-users distribution in the investigated scenario, a preliminary analysis on the joint application of users grouping algorithms with multi-antenna and multi-user techniques has been performed. In conclusion, the importance and impact of other fundamental physical layer operations, such as channel estimation and power control, on the overall end-to-end system behavior and performance were highlighted.

RNA interference technology as a potential control method for fruit and horticultural crops pathogens Botrytis cinerea and Plasmopara viticola

Chapter 1, a general introduction on Botrytis cinerea and its threat to crop production is presented. What Botrytis looks like, its life cycle, why it is a threat to agricultural production, its worldwide pest status, and its current state of management is further elaborated on. Chapter 2, a general introduction on Plasmopara viticola, its threat to grape production and management strategies presented. Chapter 3, titled " RNA Interference Strategies for Future Management of Plant Pathogenic Fungi: Prospects and Challenges ", presents the rapid improvement and extensive implementation of RNA interference (RNAi) technology for the management of fungal pathogens. In this chapter, we describe the application of exogenous RNAi involved in plant pathogenic fungi and discuss dsRNA production, formulation, and RNAi delivery methods. Chapter 4, titled " Exogenous dsRNAs against chitin synthase and glucan synthase genes suppress the growth of the pathogenic fungus Botrytis cinerea " addresses two important questions: Is RNAi technology functional for B. cinerea control ? And which target genes can be exploited for RNAi-based B.cinerea disease control ? Upon target genes selections, an exogenous RNAi protocol was set up and we could effectively deliver a known dose of bacterially produced double stranded RNA (dsRNA) to induce RNAi in B. cinerea. Chapter 5, titled " Double-Stranded RNA Targeting Dicer-Like Genes Compromises the Pathogenicity of Plasmopara viticola on Grapevine “, which deals mainly on RNAi induction against Plasmopara viticola. This chapter addresses two main questions: Is RNAi technology functional in contrasting Plasmopara viticola? And which target genes can be exploited for RNAi-based disease control in Plasmopara viticola?. In the last Chapter (Chapter 6) titled “General discussions and perspectives for future research”, the major research findings from this thesis are discussed together with perspectives for future research.

Non-invasive detection of acute cell-mediated graft rejection in paediatric heart transplant recipients: the role of cardiovascular magnetic resonance

Background and Aim: Acute cardiac rejection is currently diagnosed by endomyocardial biopsy (EMB), but multiparametric cardiac magnetic resonance (CMR) may be a non-invasive alternative by its capacity for myocardial structure and function characterization. Our primary aim was to determine the utility of multiparametric CMR in identifying acute graft rejection in paediatric heart transplant recipients. The second aim was to compare textural features of parametric maps in cases of rejection versus those without rejection. Methods: Fifteen patients were prospectively enrolled for contrast-enhanced CMR followed by EMB and right heart catheterization. Images were acquired on a 1,5 Tesla scanner including T1 mapping (modified Look-Locker inversion recovery sequence – MOLLI) and T2 mapping (modified GraSE sequence). The extracellular volume (ECV) was calculated using pre- and post-gadolinium T1 times of blood and myocardium and the patient’s hematocrit. Markers of graft dysfunction including hemodynamic measurements from echocardiography, catheterization and CMR were collated. Patients were divided into two groups based on degree of rejection at EMB: no rejection with no change in treatment (Group A) and acute rejection requiring new therapy (Group B). Statistical analysis included student’t t test and Pearson correlation. Results: Acute rejection was diagnosed in five patients. Mean T1 values were significantly associated with acute rejection. A monotonic, increasing trend was noted in both mean and peak T1 values, with increasing degree of rejection. ECV was significantly higher in Group B. There was no difference in T2 signal between two groups. Conclusion: Multiparametric CMR serves as a noninvasive screening tool during surveillance encounters and may be used to identify those patients that may be at higher risk of rejection and therefore require further evaluation. Future and multicenter studies are necessary to confirm these results and explore whether multiparametric CMR can decrease the number of surveillance EMBs in paediatric heart transplant recipients.