Towards personalized immunotherapy : development of in vitro models for imaging natural killer cell behavior in the tumor microenvironment

Tremendous advances in the tumor immunology field have transformed immunotherapy from a promising approach to a standard clinical practice. However, a subset of cancer patients is non-responsive to immunotherapy. More research is therefore needed to understand the mechanisms underlying tumor resistance to immunotherapeutic treatments. The aim of this doctoral work was to develop new tools to study the mechanisms of cancer immunosurveillance and to test immunotherapeutic treatments in vitro. In this thesis, I describe the methods developed, and I discuss the main biological findings obtained by using these methods. The thesis is organized as follows. A short historical background of immunotherapy is provided in Chapter 1. Chapter 2 describes the principles of NK cell-mediated cancer immunosurveillance, and provides an overview on rare cancers, mainly focusing on sarcoma. The research aims are listed in Chapter 3. In Chapter 4, I describe the cell culture methods and cell analysis techniques relevant for my doctoral work. In Chapter 5, I describe the methods we developed to culture tumor spheroids in vitro using ultrasonic standing waves in microwell chips, focusing on the theory, design, and applications. Chapter 6 and Chapter 7 focus on the biological findings obtained using our platform in combination with traditional immunological methods, followed by future implementations discussed in Chapter 8. The constituent papers are provided at the end of the thesis. In Paper I, we combined the use of the microwell chip, ultrasonic standing waves and a protein-repellent polymer coating to enable the production of spheroids from multiple cell types. In absence of cell adhesion to the chip, spheroids could be collected and further analyzed by off-the-chip techniques. In Paper II, we designed a novel multichambered microwell chip to perform multiplexed fluorescence screening of two- or three-dimensional cell cultures. The platform allows the direct assessment of drug or immune cell cytotoxic efficacy, making it a promising tool for individualized cytotoxicity tests for personalized medicine. In Paper III, we investigate the function of PVR receptors in NK cells interacting with renal carcinoma spheroids, and the impact of PVR in NK cell-based cellular immunotherapy. We demonstrated that variations in PVR expression are primarily recognized by the inhibitory receptor TIGIT, while DNAM-1 strongly contributes to NK cell activation mainly through PVR-independent mechanisms. We performed NK cell-based cytotoxicity assays against renal carcinoma spheroids in the microwell chip. Anti-TIGIT treatment was effective only for TIGIThigh NK cells both when used as monotherapy or in combination with other drugs, suggesting that only a fraction of patients might respond to anti-TIGIT therapy. In Paper IV, a similar approach was used with primary sarcomas. We cultured patient-derived sarcoma spheroids and tested NK cell-based immunotherapy in the microwell chip, either alone or in combination with antibody therapy, and we identified promising treatment combinations. In Paper V, we applied the use of expansion microscopy to visualize NK cells infiltrating renal carcinoma spheroids. In conclusion, our multi-disciplinary work shows the development of new imaging-based platform and its use to study the mechanisms of NK cell-mediated tumor surveillance and for personalized therapy

Databases of surface wave dispersion

Observations of seismic surface waves provide the most important constraint on the elastic properties of the Earth’s lithosphere and upper mantle. Two databases of fundamental mode surface wave dispersion were recently compiled and published by groups at Harvard (Ekström et al., 1997) and Utrecht/Oxford (Trampert and Woodhouse, 1995, 2001), and later employed in 3-d global tomographic studies. Although based on similar sets of seismic records, the two databases show some significant discrepancies. We derive phase velocity maps from both, and compare them to quantify the discrepancies and assess the relative quality of the data; in this endeavour, we take careful account of the effects of regularization and parametrization. At short periods, where Love waves are mostly sensitive to crustal structure and thickness, we refer our comparison to a map of the Earth’s crust derived from independent data. On the assumption that second-order effects like seismic anisotropy and scattering can be neglected, we find the measurements of Ekström et al. (1997) of better quality; those of Trampert and Woodhouse (2001) result in phase velocity maps of much higher spatial frequency and, accordingly, more difficult to explain and justify geophysically. The discrepancy is partly explained by the more conservative a priori selection of data implemented by Ekström et al. (1997). Nevertheless, it becomes more significant with decreasing period, which indicates that it could also be traced to the different measurement techniques employed by the authors

Structure of the Antarctic plate by analysis of surface-wave group speed and a global Haar-wavelet model representation

The unique tectonic setting of the Antarctic plate — all surrounded by active ocean ridges, geodynamically stationary, and crossed by the largest asymmetric continental rift — make it a very interesting subject, that recently has received increased attention because of the International Polar Year (IPY). We present a seismic tomographic study targeted at improving the resolution of previous models, by virtue of an extended dataset and a multiresolution spherical-wavelet based model representation. We limit the present study to modeling laterally-varying fundamental-mode Rayleigh wave group speed, that is mainly related to the structure of crust and uppermost mantle. We use single-station dispersion analysis on the surface wave train to measure group arrival times of the Rayleigh-wave fundamental mode in the period range between 30 and 150 s. On each seismogram we iteratively apply a multiple filter – to identify group arrival times with frequency – and a phase-matched filter, to isolate the fundamental mode within the wave train. We then use dispersion measurements to compute two-dimensional maps of wave group speed in the region. We parameterize the Earth using nearly-orthogonal spherical Haar wavelets based on iterative subdivisions of the icosahedron. This representation is particularly suited to implement multi-resolution, as needed for a regional model embedded into a global one. The model shows with increased detail important features such as the narrow transition between cratonic structure in East Antarctica and accreted West Antarctica, separated by the Transantarctic Mountains bordering the rift. Inclusion of data from temporary experiments carried on within the IPY, as they become available, will locally further improve resolution

L’evoluzione dei fattori di rischio di povertà in Italia e l’impatto della crisi finanziaria

Il lavoro ha come obiettivo quello di analizzare le dinamiche dei fattori di rischio di povertà in Italia, focalizzando l'attenzione sulla liquidità delle famiglie, utilizzando tre livelli differenti di analisi. Il primo è un livello descrittivo che consiste nella costruzione delle serie storiche sull'incidenza della povertà e sulle abitudini di consumo delle famiglie, individuando i fattori di rischio di povertà tra le variabili socio-demografiche. Il secondo livello di analisi è la costruzione di un modello econometrico sui comportamenti di consumo delle famiglie in funzione della liquidità, facendo riferimento a uno studio relativo al mercato degli Stati Uniti, che individua le famiglie wealthy hand-to-mouth come famiglie soggette alla vulnerabilità alla povertà a causa di un investimento che ne ha limitato la liquidità. Il terzo livello è l'analisi del rischio di cadere in condizione di povertà, nel periodo delle due più recenti crisi, utilizzando la latent transition analysis, un modello a classi latenti che permette di stimare una variabile multidimensionale con più stati latenti e la probabilità di cambiare lo stato latente da un periodo all'altro. In tal senso, il modello permette di analizzare la vulnerabilità, intesa come la probabilità di cadere in condizione di povertà nel periodo successivo, tenendo conto dei risultati delle serie storiche e del modello econometrico

Effects of Uncertainty on Household Saving Rate

This PhD thesis attempts to investigate the role of economic uncertainty in driving the behaviour of household savings for six European countries. Focusing on three main sources of economic uncertainty Unemployment Risk, Fiscal Policy Uncertainty and Financial Crisis-Investment risk, I construct a Structural Vector Autoregressive (SVAR) model comprising of the Household Saving Rate, main variable of interest; the unemployment rate, to proxy labour income uncertainty and the risk of an income loss; the volatility of financial stock prices per each country, to detect for the presence of financial uncertainty/crisis; a policy uncertainty indicator, using alternatively the Policy Uncertainty Index devised by Baker, Bloom, and Davis (2012), the Debt to GDP ratio or the Government Surplus/Deficit to GDP ratio. A comparison among country-specific cumulative impulse response functions suggests that: 1. Household saving rate’s response to a change in investment risk is ambiguous, due to two counterbalancing effects: higher risk increases the volatility of future consumption and thus stimulates the accumulation of savings, while a more uncertain rate of return reduces the attractiveness of saving since it increases the risk of capital losses. 2. A labour uncertainty shock is detrimental or a booster for saving depending on whether the downward pressures on saving rate due to lower saving from unemployed people, prevails or not over the higher households propensity to save for precautionary reasons. 3. Fiscal policy instruments and related uncertainty influence the savings pattern of the private sector: private saving falls when governments reduce deficits (or the debt level) or run large budget surpluses and vice versa, as suggested by the Ricardian paradigm. I then propose another possible approach to the analysis, a Bayesian estimation of the reduced form VARs for the panel of European countries, as a Hierarchical Linear Model, with the future aim of improving estimation results

Insediamenti conventuali nei primi anni del XIV secolo nella Civitas Sanctae Mariae, odierna Lucera

In August 1300, Lucera was subjected to a “depopulation” of the Muslim inhabitants with the concurrent destruction of the buildings symbolising their faith. A new cathedral was built and the churches of four convents of Luceria Saracenorum in the Civitas Sanctae Mariae were transformed. Indeed, the urban layout of these churches seems to correspond to a precise will, that is, they seem to be arranged on the sides of a square circumscribed by the walls, at the centre of which is the cathedral