Cell death mechanisms triggered by monoclonal antibodies against CD99 in Ewing sarcoma: Cross-talk between MDM2, IGF-1R and Ras/MAPK

CD99 is a 32 kDa transmembrane protein whose high expression characterizes Ewing sarcoma (ES), a very aggressive pediatric bone tumor. In addition to its diagnostic value, CD99 has therapeutic potential since it leads to rapid and massive ES cell death when engaged with specific antibodies. Here a novel mechanism of cell death triggered via CD99 is shown, leading, ultimately, to the appearance of macropinocytotic vescicles. Anti-CD99 mAb 0662 induces MDM2 ubiquitination and degradation, which causes not only a p53 reactivation but also the IGF-1R induction and its subsequent internalization; CD99 results internalized together with IGF-1R inside endosomes, but then the two molecules display a different sorting: CD99 is degraded, while IGF-1R is recycled on the surface, causing, as a final step, the up-regulation of RAS-MAPK. High-expressing CD99 mesenchymal stem cells show mild Ras induction but no p53 activation and escape cell death, but in presence of EWS/FLI1 mesenchymal stem cells expressing CD99 show a stronger Ras induction and a p53 reactivation, leading to a significant cell death rate. We propose that CD99 triggering in a EWS/FLI1-driven oncogenetic context creates a synergy between RAS upregulation and p53 activation in ES cells, leading to cell death. Moreover, our data rule out possible concerns on toxicity related to the broad CD99 expression in normal tissues and provide the rationale for the therapeutic use of anti-CD99 MAbs in the clinic.

IGF system, CD99 and tumor-specific chromosomal translocations: evaluation of their cross-talk and definition of their role in Ewing sarcoma and prostate cancer

Aberrant expression of ETS transcription factors, including FLI1 and ERG, due to chromosomal translocations has been described as a driver event in initiation and progression of different tumors. In this study, the impact of prostate cancer (PCa) fusion gene TMPRSS2-ERG was evaluated on components of the insulin-like growth factor (IGF) system and the CD99 molecule, two well documented targets of EWS-FLI1, the hallmark of Ewing sarcoma (ES). The aim of this study was to identify common or distinctive ETS-related mechanisms which could be exploited at biological and clinical level. The results demonstrate that IGF-1R represents a common target of ETS rearrangements as ERG and FLI1 bind IGF-1R gene promoter and their modulation causes alteration in IGF-1R protein levels. At clinical level, this mechanism provides basis for a more rationale use of anti-IGF-1R inhibitors as PCa cells expressing the fusion gene better respond to anti-IGF-1R agents. EWS-FLI1/IGF-1R axis provides rationale for combination of anti-IGF-1R agents with trabectedin, an alkylator agent causing enhanced EWS-FLI1 occupancy on the IGF-1R promoter. TMPRSS2-ERG also influences prognosis relevance of IGF system as high IGF-1R correlates with a better biochemical progression free survival (BPFS) in PCa patients negative for the fusion gene while marginal or no association was found in the total cases or TMPRSS2-ERG-positive cases, respectively. This study indicates CD99 is differentially regulated between ETS-related tumors as CD99 is not a target of ERG. In PCa, CD99 did not show differential expression between TMPRSS2-ERG-positive and –negative cells. A direct correlation was anyway found between ERG and CD99 proteins both in vitro and in patients putatively suggesting that ERG target genes comprehend regulators of CD99. Despite a little trend suggesting a correlation between CD99 expression and a better BPFS, no clinical relevance for CD99 was found in the field of prognostic biomarkers.

Self-Organizing Mechanisms for Task Allocation in a Knowledge-Based Economy

A prevalent claim is that we are in knowledge economy. When we talk about knowledge economy, we generally mean the concept of “Knowledge-based economy” indicating the use of knowledge and technologies to produce economic benefits. Hence knowledge is both tool and raw material (people’s skill) for producing some kind of product or service. In this kind of environment economic organization is undergoing several changes. For example authority relations are less important, legal and ownership-based definitions of the boundaries of the firm are becoming irrelevant and there are only few constraints on the set of coordination mechanisms. Hence what characterises a knowledge economy is the growing importance of human capital in productive processes (Foss, 2005) and the increasing knowledge intensity of jobs (Hodgson, 1999). Economic processes are also highly intertwined with social processes: they are likely to be informal and reciprocal rather than formal and negotiated. Another important point is also the problem of the division of labor: as economic activity becomes mainly intellectual and requires the integration of specific and idiosyncratic skills, the task of dividing the job and assigning it to the most appropriate individuals becomes arduous, a “supervisory problem” (Hogdson, 1999) emerges and traditional hierarchical control may result increasingly ineffective. Not only specificity of know how makes it awkward to monitor the execution of tasks, more importantly, top-down integration of skills may be difficult because ‘the nominal supervisors will not know the best way of doing the job – or even the precise purpose of the specialist job itself – and the worker will know better’ (Hogdson,1999). We, therefore, expect that the organization of the economic activity of specialists should be, at least partially, self-organized. The aim of this thesis is to bridge studies from computer science and in particular from Peer-to-Peer Networks (P2P) to organization theories. We think that the P2P paradigm well fits with organization problems related to all those situation in which a central authority is not possible. We believe that P2P Networks show a number of characteristics similar to firms working in a knowledge-based economy and hence that the methodology used for studying P2P Networks can be applied to organization studies. Three are the main characteristics we think P2P have in common with firms involved in knowledge economy: - Decentralization: in a pure P2P system every peer is an equal participant, there is no central authority governing the actions of the single peers; - Cost of ownership: P2P computing implies shared ownership reducing the cost of owing the systems and the content, and the cost of maintaining them; - Self-Organization: it refers to the process in a system leading to the emergence of global order within the system without the presence of another system dictating this order. These characteristics are present also in the kind of firm that we try to address and that’ why we have shifted the techniques we adopted for studies in computer science (Marcozzi et al., 2005; Hales et al., 2007 [39]) to management science.

Cross-Talk tra Bifidobacterium e intestino umano: impatto sulle attività health promoting

Bifidobacteria constitute up to 3% of the total microbiota and represent one of the most important healthpromoting bacterial groups of the human intestinal microflora. The presence of Bifidobacterium in the human gastrointestinal tract has been directly related to several health-promoting activities; however, to date, no information about the specific mechanisms of interaction with the host is available. The first health-promoting activities studied in these job was the oxalate-degrading activity. Oxalic acid occurs extensively in nature and plays diverse roles, especially in pathological processes. Due to its highly oxidizing effects, hyper absorption or abnormal synthesis of oxalate can cause serious acute disorders in mammals and be lethal in extreme cases. Intestinal oxalate-degrading bacteria could therefore be pivotal in maintaining oxalate homeostasis, reducing the risk of kidney stone development. In this study, the oxalate-degrading activity of 14 bifidobacterial strains was measured by a capillary electrophoresis technique. The oxc gene, encoding oxalyl-CoA decarboxylase, a key enzyme in oxalate catabolism, was isolated by probing a genomic library of B. animalis subsp. lactis BI07, which was one of the most active strains in the preliminary screening. The genetic and transcriptional organization of oxc flanking regions was determined, unravelling the presence of other two independently transcribed open reading frames, potentially responsible for B. animalis subsp. lactis ability to degrade oxalate. Transcriptional analysis, using real-time quantitative reverse transcription PCR, revealed that these genes were highly induced in cells first adapted to subinhibitory concentrations of oxalate and then exposed to pH 4.5. Acidic conditions were also a prerequisite for a significant oxalate degradation rate, which dramatically increased in oxalate pre-adapted cells, as demonstrated in fermentation experiments with different pH-controlled batch cultures. These findings provide new insights in the characterization of oxalate-degrading probiotic bacteria and may support the use of B. animalis subsp. lactis as a promising adjunct for the prophylaxis and management of oxalate-related kidney disease. In order to provide some insight into the molecular mechanisms involved in the interaction with the host, in the second part of the job, we investigated whether Bifidobacterium was able to capture human plasminogen on the cell surface. The binding of human plasminogen to Bifidobacterium was dependent on lysine residues of surface protein receptors. By using a proteomic approach, we identified six putative plasminogen-binding proteins in the cell wall fraction of three strain of Bifidobacterium. The data suggest that plasminogen binding to Bifidobactrium is due to the concerted action of a number of proteins located on the bacterial cell surface, some of which are highly conserved cytoplasmic proteins which have other essential cellular functions. Our findings represent a step forward in understanding the mechanisms involved in the Bifidobacterium-host interaction. In these job w studied a new approach based on to MALDI-TOF MS to measure the interaction between entire bacterial cells and host molecular target. MALDI-TOF (Matrix Assisted Laser Desorption Ionization-Time of Flight)—mass spectrometry has been applied, for the first time, in the investigation of whole Bifidobacterium cells-host target proteins interaction. In particular, by means of this technique, a dose dependent human plasminogen-binding activity has been shown for Bifidobacterium. The involvement of lysine binding sites on the bacterial cell surface has been proved. The obtained result was found to be consistent with that from well-established standard methodologies, thus the proposed MALDI-TOF approach has the potential to enter as a fast alternative method in the field of biorecognition studies involving in bacterial cells and proteins of human origin.

Role of caveolin-1 in the proliferation of solid tumours in vitro

Caveolin-1 (Cav-1), the essential structural constituent of caveolae, which are flask-shaped invaginations of the plasma membrane, has been found to play a key role in the modulation of cell proliferation and cancer development. It seems to act as an oncosuppressor or a promoter of growth, depending on the histotype, stage and grade of each tumour. The aim of this study was to analyze the effects of Caveolin-1 gene silencing on the proliferation of human lung cancer and osteosarcoma in vitro. Our data show that Cav-1 silencing blocks the growth in both metastatic lung cancer cell lines analyzed, suggesting a proliferation promoting action of the protein in these cells. A marked decrease of phospho-Akt, phospho-ERK, STAT3, cyclin D1, CDK4 and consequently of phospho-Rb expression was evident in the cells treated with Cav-1 siRNA. With regards to osteosarcoma, we demonstrated that the suppression of Cav-1 results in the blocking of MG-63 and in the slowing down of HOS proliferation, suggesting a role for Cav-1 as a promoter of tumour growth in these cell lines. A marked decrease of phospho-Akt, cyclin E, CDK2 and phospho-Rb and an increase of p21 expression levels were evident in the cells treated with Cav-1 siRNA. Our results suggest two new cell cycle inhibiting pathways, mediated by Cav-1 knock-down, and provide new insights into the molecular mechanisms underlying the tumour-promoting role of Cav-1 in lung cancer and osteosarcoma. In this work we also investigated the role of estrogens in lung cancer and the functional cross-talk between Cav-1 and estrogens/estrogen receptors in it. Our results show that 17β-estradiol induces proliferation either in RAL or in SCLC-R1 cells and that both cell lines are sensitive to 4-OHT antiproliferative effect. The sensitivity to estrogen stimulation seems to be gender- and/or histological type-independent in metastatic lung cancer in vitro.

Europa e Catasto

We study first the different cadastral systems in the EU countries and their perspective in the European Law context, especially in their tax law aspect and about the different building tax system. We talk about the most important aspect, taxation, and the European Unionʼs influence, particularly the European Court. But not only speak about the influence in the Member stateʼs building tax, also find another influences ways, with some European policies. All these aspects and another show a tendency to the cadastral integration, not direct, but existent in the indirect way. About other aspects, the study holds the dual nature of the cadastre, social (like social science), and their tax aspect, and technic nature. The Inspire information net can generate a new way to the tax information exchange between European countries. The investigation end with a comparison of the different cadastral systems in EU countries, and about the edification tax law too. This report holds the tax nature of the cadastre, the need to be considered like social-technic complex. Diverse international organization consider that is a multipurpose instrument and institution, but seem to forget their original purpose, their tax purpose, that was the central aspect in their origin and that don can`t be forget in the new world that raises after the world financial crisis.

Airway Basal Cell Vascular Endothelial Growth Factor-mediated Cross-Talk Regulates Endothelial Cell Dependent Growth Support of Human Airway Basal Cells

The human airway epithelium is a pseudostratified heterogenous layer comprised of cili-ated, secretory, intermediate and basal cells. As the stem/progenitor population of the airway epi-thelium, airway basal cells differentiate into ciliated and secretory cells to replenish the airway epithelium during physiological turnover and repair. Transcriptome analysis of airway basal cells revealed high expression of vascular endothelial growth factor A (VEGFA), a gene not typically associated with the function of this cell type. Using cultures of primary human airway basal cells, we demonstrate that basal cells express all of the 3 major isoforms of VEGFA (121, 165 and 189) but lack functional expression of the classical VEGFA receptors VEGFR1 and VEGFR2. The VEGFA is actively secreted by basal cells and while it appears to have no direct autocrine function on basal cell growth and proliferation, it functions in a paracrine manner to activate MAPK signaling cascades in endothelium via VEGFR2 dependent signaling pathways. Using a cytokine- and serum-free co-culture system of primary human airway basal cells and human endothelial cells revealed that basal cell secreted VEGFA activated endothelium to ex-press mediators that, in turn, stimulate and support basal cell proliferation and growth. These data demonstrate novel VEGFA mediated cross-talk between airway basal cells and endothe-lium, the purpose of which is to modulate endothelial activation and in turn stimulate and sustain basal cell growth.

Molecular and functional characterization of the interplay between malignant and stromal cells in acute myeloid leukemia and myelodysplastic syndrome

In this thesis, we studied the cross-talk between malignant cells and stromal cells, with the aim to elucidate the respective contribution to myeloid neoplasm onset and progression. First, we characterized and compared mesenchymal stromal cells (MSCs) isolated from myelodysplastic syndrome (MDS-MSCs) and acute myeloid leukemia (AML-MSCs) patients. We demonstrated that, despite some unaltered functions, patient-derived MSCs show also intrinsic, distinct functional abnormalities, which could all potentially favor a leukemia-protective bone marrow (BM) niche in vivo. Second, we investigated the ability of AML cells to modulate the AML-MSC functions. In a GEP-screening, we found that 40% of BM-derived AML samples show a higher IFN-γ expression, compared to the mean IFN-γ expression in healthy BM-derived cells. We demonstrated that in co-culture experiments, IFN-γ+ AML cells modify AML-MSC gene expression and function, inducing the up-regulation of IDO1, and consequently the generation of T regulatory cells. Finally, we wondered if the transcriptome of stromal cells could be influenced by the hematopoietic-specific alterations, i.e. Dnmt3a and Asxl1 mutations, which occur early in MDS/AML patients. We found that Dnmt3a- and Asxl1-null BM cells, when transplanted in wild-type mice, induce profound and deletion-specific modifications in the transcriptome of wild-type BM stromal cells, suggesting the ability of Dnmt3a- and Asxl1-null BM cells to shape the niche. Furthermore, we compared the transcriptome of wild-type BM stromal cells, obtained from transplantation experiments, with that of MSCs isolated from low-risk MDS patients with DNMT3A and ASXL1 mutations, and we highlighted some common modifications, which could be potentially relevant for human disease and specific for DNMT3A/ASXL1 mutations. In conclusion, this thesis pointed out that there is a bi-directional cross-talk, in which stromal cells can influence malignant cells, and in turn malignant/pre-malignant cells can alter stromal cell gene expression and function. Both mechanisms could potentially contribute to the pathogenesis of myeloid malignancies.

Essays on Information Economics

This thesis consists of three essays on information economics. I explore how information is strategically communicated or designed by senders who aim to influence the decisions of a receiver. In the first chapter, I study a cheap talk game between two imperfectly informed experts and a decision maker. The experts receive noisy signals about the state and sequentially communicate the relevant information to the decision maker. I refine the self-serving belief system under uncertainty and Ι characterise the most informative equilibrium that might arise in such environments.In the second chapter, I consider the case where a decision maker seeks advice from a biased expert who cares also about establishing a reputation of being competent. The expert has the incentives to misreport her information but she faces a trade-off between the gain from misrepresentation and the potential reputation loss. I show that the equilibrium is fully-revealing if the expert is not too biased and not too highly reputable. If there is competition between two experts the information transmission is always improved. However, in cases where the experts are more than two the result is ambiguous, and it depends on the players’ prior belief over states.In the last chapter, I consider a model of strategic communication where a privately and imperfectly informed sender can persuade a receiver. The sender may receive favorable or unfavorable private information about her preferred state. I describe two ways that are adopted in real life situations and theoretically improve equilibrium informativeness given sender's private information. First, a policy that suggests symmetry constraints to the experiments' choice. Second, an approval strategy characterised by a low precision threshold where the receiver will accept the sender with a positive probability and a higher one where the sender will be accepted with certainty.