In vitro characterisation and expansion of human regulatory T cells for their in vivo application in the induction of tolerance in haematopoietic stem cell and solid organ transplantation

Solid organ transplantation (SOT) is considered the treatment of choice for many end-stage organ diseases. Thus far, short term results are excellent, with patient survival rates greater than 90% one year post-surgery, but there are several problems with the long term acceptance and use of immunosuppressive drugs. Hematopoietic Stem Cells Transplantation (HSCT) concerns the infusion of haematopoietic stem cells to re-establish acquired and congenital disorders of the hematopoietic system. The main side effect is the Graft versus Host Disease (GvHD) where donor T cells can cause pathology involving the damage of host tissues. Patients undergoing acute or chronic GvHD receive immunosuppressive regimen that is responsible for several side effects. The use of immunosuppressive drugs in the setting of SOT and GvHD has markedly reduced the incidence of acute rejection and the tissue damage in GvHD however, the numerous adverse side effects observed boost the development of alternative strategies to improve the long-term outcome. To this effect, the use of CD4+CD25+FOXP3+ regulatory T cells (Treg) as a cellular therapy is an attractive approach for autoimmunity disease, GvHD and limiting immune responses to allograft after transplantation. Treg have a pivotal role in maintaining peripheral immunological tolerance, by preventing autoimmunity and chronic inflammation. Results of my thesis provide the characterization and cell processing of Tregs from healthy controls and patients in waiting list for liver transplantation, followed by the development of an efficient expansion-protocol and the investigation of the impact of the main immunosuppressive drugs on viability, proliferative capacity and function of expanded cells after expansion. The conclusion is that ex vivo expansion is necessary to infuse a high Treg dose and although many other factors in vivo can contribute to the success of Treg therapy, the infusion of Tregs during the administration of the highest dose of immunosuppressants should be carefully considered.

Detailed study of the seismotectonic setting in the Lucanian Apennines and surrounding areas (Southern Italy)

In this research work I analyzed the instrumental seismicity of Southern Italy in the area including the Lucanian Apennines and Bradano foredeep, making use of the most recent seismological database available so far. I examined the seismicity occurred during the period between 2001 and 2006, considering 514 events with magnitudes M ≥ 2.0. In the first part of the work, P- and S-wave arrival times, recorded by the Italian National Seismic Network (RSNC) operated by the Istituto Nazionale di Geofisica e Vulcanologia (INGV), were re-picked along with those of the SAPTEX temporary array (2001–2004). For some events located in the Upper Val d'Agri, I also used data from the Eni-Agip oil company seismic network. I computed the VP/VS ratio obtaining a value of 1.83 and I carried out an analysis for the one-dimensional (1D) velocity model that approximates the seismic structure of the study area. After this preliminary analysis, making use of the records obtained in the SeSCAL experiment, I incremented the database by handpicking new arrival times. My final dataset consists of 15,666 P- and 9228 S-arrival times associated to 1047 earthquakes with magnitude ML ≥ 1.5. I computed 162 fault-plane solutions and composite focal mechanisms for closely located events. I investigated stress field orientation inverting focal mechanism belonging to the Lucanian Apennine and the Pollino Range, both areas characterized by more concentrated background seismicity. Moreover, I applied the double difference technique (DD) to improve the earthquake locations. Considering these results and different datasets available in the literature, I carried out a detailed analysis of single sub-areas and of a swarm (November 2008) recorded by SeSCAL array. The relocated seismicity appears more concentrated within the upper crust and it is mostly clustered along the Lucanian Apennine chain. In particular, two well-defined clusters were located in the Potentino and in the Abriola-Pietrapertosa sector (central Lucanian region). Their hypocentral depths are slightly deeper than those observed beneath the chain. I suggest that these two seismic features are representative of the transition from the inner portion of the chain with NE-SW extension to the external margin characterized by dextral strike-slip kinematics. In the easternmost part of the study area, below the Bradano foredeep and the Apulia foreland, the seismicity is generally deeper and more scattered and is associated to the Murge uplift and to the small structures present in the area. I also observed a small structure NE-SW oriented in the Abriola-Pietrapertosa area (activated with a swarm in November 2008) that could be considered to act as a barrier to the propagation of a potential rupture of an active NW-SE striking faults system. Focal mechanisms computed in this study are in large part normal and strike-slip solutions and their tensional axes (T-axes) have a generalized NE-SW orientation. Thanks to denser coverage of seismic stations and the detailed analysis, this study is a further contribution to the comprehension of the seismogenesis and state of stress of the Southern Apennines region, giving important contributions to seismotectonic zoning and seismic hazard assessment.

Evaluation of Antitumoral activity of bone targeted drugs/conventional chemotherapies and identification of biomarkers for the selection of patients with breast cancer for the bone targeted therapy in adjuvant setting

Bone metastases are responsible for different clinical complications defined as skeletal-related events (SREs) such as pathologic fractures, spinal cord compression, hypercalcaemia, bone marrow infiltration and severe bone pain requiring palliative radiotherapy. The general aim of these three years research period was to improve the management of patients with bone metastases through two different approaches of translational research. Firstly in vitro preclinical tests were conducted on breast cancer cells and on indirect co-colture of cancer cells and osteoclasts to evaluate bone targeted therapy singly and in combination with conventional chemotherapy. The study suggests that zoledronic acid has an antitumor activity in breast cancer cell lines. Its mechanism of action involves the decrease of RAS and RHO, as in osteoclasts. Repeated treatment enhances antitumor activity compared to non-repeated treatment. Furthermore the combination Zoledronic Acid + Cisplatin induced a high antitumoral activity in the two triple-negative lines MDA-MB-231 and BRC-230. The p21, pMAPK and m-TOR pathways were regulated by this combined treatment, particularly at lower Cisplatin doses. A co-colture system to test the activity of bone-targeted molecules on monocytes-breast conditioned by breast cancer cells was also developed. Another important criticism of the treatment of breast cancer patients, is the selection of patients who will benefit of bone targeted therapy in the adjuvant setting. A retrospective case-control study on breast cancer patients to find new predictive markers of bone metastases in the primary tumors was performed. Eight markers were evaluated and TFF1 and CXCR4 were found to discriminate between patients with relapse to bone respect to patients with no evidence of disease. In particular TFF1 was the most accurate marker reaching a sensitivity of 63% and a specificity of 79%. This marker could be a useful tool for clinicians to select patients who could benefit for bone targeted therapy in adjuvant setting.