Exploring novel targeted therapeutic strategies against Acute Myeloid Leukemia cells based on inhibition of bromodomain proteins and CDC20 activity

Acute myeloid leukemia (AML) is a haematological malignancies arising from the accumulation of undifferentiated myeloid progenitors with an uncontrolled proliferation. The genomic landscape of AML revealed that the disease is characterized by high level of heterogeneity and is subjected to clonal evolution driven by selective pressure of chemotherapy. In this study, we investigated the therapeutic effects of the inhibition of BRD4 and CDC20 in vitro and ex vivo. We demonstrated that inhibition of BRD4 with GSK1215101A in AML cell lines was effective under hypoxia. It induced the activation of antioxidant response both, at transcriptomic and metabolomic levels, driven by enrichment of NRF2 pathway under normoxic and hypoxic condition. Moreover, the combined treatment with Omaveloxolone, a drug inducing NRF2 activation and NF-κB inhibition, potentiated the effects on apoptosis and colony forming capacity of stem progenitor cells. Lastly, gene expression profiling data revealed that combination treatment induced major changes in genes related to cell cycle, together with enrichment of cell differentiation pathways and negative regulation of WNT, in normoxia and hypoxia. Regarding CDC20, we observed its up-regulation in AML patients. Treatment with two different inhibitors, Apcin and proTAME, was effective in primary AML cells and in AML cell lines, through induction of apoptosis and mitotic arrest. The lack of correlation between proliferation markers and CDC20 levels in AML cell subpopulations supports the idea of alternative CDC20 functions, independent from its essential role during mitosis. CDC20-KD experiments conducted in AML cell lines revealed a mild effect on apoptosis induction, but no significant change in cell cycle progression. In summary, these results allowed the identification of a new strategy combination to improve the effects of BRD4 inhibition on LSC residing in the BM hypoxic niche, and provide some new evidence regarding the potential role of CDC20 as a new target for AML treatment.

Data protection by design in the e-health care sector: theoretical and applied perspectives

In the digital age, e-health technologies play a pivotal role in the processing of medical information. As personal health data represents sensitive information concerning a data subject, enhancing data protection and security of systems and practices has become a primary concern. In recent years, there has been an increasing interest in the concept of Privacy by Design, which aims at developing a product or a service in a way that it supports privacy principles and rules. In the EU, Article 25 of the General Data Protection Regulation provides a binding obligation of implementing Data Protection by Design technical and organisational measures. This thesis explores how an e-health system could be developed and how data processing activities could be carried out to apply data protection principles and requirements from the design stage. The research attempts to bridge the gap between the legal and technical disciplines on DPbD by providing a set of guidelines for the implementation of the principle. The work is based on literature review, legal and comparative analysis, and investigation of the existing technical solutions and engineering methodologies. The work can be differentiated by theoretical and applied perspectives. First, it critically conducts a legal analysis on the principle of PbD and it studies the DPbD legal obligation and the related provisions. Later, the research contextualises the rule in the health care field by investigating the applicable legal framework for personal health data processing. Moreover, the research focuses on the US legal system by conducting a comparative analysis. Adopting an applied perspective, the research investigates the existing technical methodologies and tools to design data protection and it proposes a set of comprehensive DPbD organisational and technical guidelines for a crucial case study, that is an Electronic Health Record system.

Better Regulation in Latin American Countries: A tool for accountability?

In the last few years, a steadily increasing number of Latin American countries have been adopting policy assessment instruments and new governance structures for them, as part of their policymaking process. Even though the literature argues that these instruments serve, among other things, as tools for accountability, for this to be so, it is necessary to take into account the legal system, decision-making process, and regulatory relationships that exist in the adopting countries. This Thesis researches the policy assessment arrangements adopted and implemented in the Latin American region to understand why are these countries adopting and implementing tools for policy evaluation? Can this contribute towards regulatory accountability, and if so, in which conditions? The Thesis first analyzes the rationales that these countries might have to adopt these regulatory policy arrangements. It then studies the various tools used for policy assessment, paying attention to the scope of the assessments, the times and the stages on which regulations are assessed, referred to as the Policy Evaluation Cycle (PEC), as well as to the governance of these processes. The Thesis develops a framework where each of these components are organized and classified based on which goals or rationales they serve. This can assist countries on deciding how to implement their policy evaluation arrangements, to serve their own goals. Since all of the studied countries have presidential systems, this Thesis studies how regulations are made in this system, and the multiple needed delegations for policymaking, which results in various regulatory relationships. Thus, the desired accountability of policymakers towards their different forums makes relevant the adoption this agenda for regulatory accountability reasons. Bringing to together the literatures on public law, accountability and policy evaluation, this Thesis builds a framework for assessing the contribution towards accountability that each stage of the PEC might have in a specific regulatory relationship. The framework shows to which degree the stages, and the cycle as a whole, contribute towards accountability in specific relationships of a presidential constitutional system. The results evidence that even when a policy assessment structure might contribute towards accountability, this contribution is not absolute as it only operates in specific regulatory relationships, and even more, only in some stages of the PEC contributing at different degrees. This framework can be used by governments or regulatory agencies as an instrument to assess the contribution to accountability of their existing or potential regulatory policy structures in order to improve it.

Greening the city: an ecosystem-based framework to support planning towards urban sustainability and resilience

Cities are small-scale complex socio-ecological systems, that host around 60% of world population. Ecosystem Services (ES) provided by urban ecosystems offer multiple benefits necessary to cope with present and future urban challenges. These ES include microclimate regulation, runoff control, as well as opportunities for mental and physical recreation, affecting citizen’s health and wellbeing. Creating a balance between urban development, land take containment, climate adaptation and availability of Urban Green Areas and their related benefits, can improve the quality of the lives of the inhabitants, the economic performance of the city and the social justice and cohesion aspects. This work starts analysing current literature around the topic of Ecosystem Services (ES), Green and Blue Infrastructure (GBI) and Nature-based Solutions (NBS) and their integration within current European and International sustainability policies. Then, the thesis focuses on the role of ES, GBI and NBS towards urban sustainability and resilience setting the basis to build the core methodological and conceptual approach of this work. The developed ES-based conceptual approach provides guidance on how to map and assess ES, to better inform policy making and to give the proper value to ES within urban context. The proposed interdisciplinary approach navigates the topic of mapping and assessing ES benefits in terms of regulatory services, with a focus on climate mitigation and adaptation, and cultural services, to enhance wellbeing and justice in urban areas. Last, this thesis proposes a trans-disciplinary and participatory approach to build resilience over time around all relevant urban ES. The two case studies that will be presented in this dissertation, the city of Bologna and the city of Barcelona, have been used to implement, tailor and test the proposed conceptual framework, raising valuable inputs for planning, policies and science.

Distributed ledger technologies between anonymity and transparency: AML/CFT regulation of cryptocurrency ecosystems in the EU

The advent of Bitcoin suggested a disintermediated economy in which Internet users can take part directly. The conceptual disruption brought about by this Internet of Money (IoM) mirrors the cross-industry impacts of blockchain and distributed ledger technologies (DLTs). While related instances of non-centralisation thwart regulatory efforts to establish accountability, in the financial domain further challenges arise from the presence in the IoM of two seemingly opposing traits: anonymity and transparency. Indeed, DLTs are often described as architecturally transparent, but the perceived level of anonymity of cryptocurrency transfers fuels fears of illicit exploitation. This is a primary concern for the framework to prevent money laundering and the financing of terrorism and proliferation (AML/CFT/CPF), and a top priority both globally and at the EU level. Nevertheless, the anonymous and transparent features of the IoM are far from clear-cut, and the same is true for its levels of disintermediation and non-centralisation. Almost fifteen years after the first Bitcoin transaction, the IoM today comprises a diverse set of socio-technical ecosystems. Building on an analysis of their phenomenology, this dissertation shows how there is more to their traits of anonymity and transparency than it may seem, and how these features range across a spectrum of combinations and degrees. In this context, trade-offs can be evaluated by referring to techno-legal benchmarks, established through socio-technical assessments grounded on teleological interpretation. Against this backdrop, this work provides framework-level recommendations for the EU to respond to the twofold nature of the IoM legitimately and effectively. The methodology cherishes the mutual interaction between regulation and technology when drafting regulation whose compliance can be eased by design. This approach mitigates the risk of overfitting in a fast-changing environment, while acknowledging specificities in compliance with the risk-based approach that sits at the core of the AML/CFT/CPF regime.

Big data and AI applications for health and research. Co-regulation mechanisms as a prosed solution for data protection law issues

Big data and AI are paving the way to promising scenarios in clinical practice and research. However, the use of such technologies might clash with GDPR requirements. Today, two forces are driving the EU policies in this domain. The first is the necessity to protect individuals’ safety and fundamental rights. The second is to incentivize the deployment of innovative technologies. The first objective is pursued by legislative acts such as the GDPR or the AIA, the second is supported by the new data strategy recently launched by the European Commission. Against this background, the thesis analyses the issue of GDPR compliance when big data and AI systems are implemented in the health domain. The thesis focuses on the use of co-regulatory tools for compliance with the GDPR. This work argues that there are two level of co-regulation in the EU legal system. The first, more general, is the approach pursued by the EU legislator when shaping legislative measures that deal with fast-evolving technologies. The GDPR can be deemed a co-regulatory solution since it mainly introduces general requirements, which implementation shall then be interpretated by the addressee of the law following a risk-based approach. This approach, although useful is costly and sometimes burdensome for organisations. The second co-regulatory level is represented by specific co-regulatory tools, such as code of conduct and certification mechanisms. These tools are meant to guide and support the interpretation effort of the addressee of the law. The thesis argues that the lack of co-regulatory tools which are supposed to implement data protection law in specific situations could be an obstacle to the deployment of innovative solutions in complex scenario such as the health ecosystem. The thesis advances hypothesis on theoretical level about the reasons of such a lack of co-regulatory solutions.

Graphene-based bioelectronic interfaces and devices and interpretative models targeting glial cells

Bioelectronic interfaces have significantly advanced in recent years, offering potential treatments for vision impairments, spinal cord injuries, and neurodegenerative diseases. However, the classical neurocentric vision drives the technological development toward neurons. Emerging evidence highlights the critical role of glial cells in the nervous system. Among them, astrocytes significantly influence neuronal networks throughout life and are implicated in several neuropathological states. Although they are incapable to fire action potentials, astrocytes communicate through diverse calcium (Ca2+) signalling pathways, crucial for cognitive functions and brain blood flow regulation. Current bioelectronic devices are primarily designed to interface neurons and are unsuitable for studying astrocytes. Graphene, with its unique electrical, mechanical and biocompatibility properties, has emerged as a promising neural interface material. However, its use as electrode interface to modulate astrocyte functionality remains unexplored. The aim of this PhD work was to exploit Graphene-oxide (GO) and reduced GO (rGO)-coated electrodes to control Ca2+ signalling in astrocytes by electrical stimulation. We discovered that distinct Ca2+dynamics in astrocytes can be evoked, in vitro and in brain slices, depending on the conductive/insulating properties of rGO/GO electrodes. Stimulation by rGO electrodes induces intracellular Ca2+ response with sharp peaks of oscillations (“P-type”), exclusively due to Ca2+ release from intracellular stores. Conversely, astrocytes stimulated by GO electrodes show slower and sustained Ca2+ response (“S-type”), largely mediated by external Ca2+ influx through specific ion channels. Astrocytes respond faster than neurons and activate distinct G-Protein Coupled Receptor intracellular signalling pathways. We propose a resistive/insulating model, hypothesizing that the different conductivity of the substrate influences the electric field at the cell/electrolyte or cell/material interfaces, favouring, respectively, the Ca2+ release from intracellular stores or the extracellular Ca2+ influx. This research provides a simple tool to selectively control distinct Ca2+ signals in brain astrocytes in neuroscience and bioelectronic medicine.