Insulin and TOR pathways regulate cellular and organismal growth through the myc oncogene in Drosophila

A large body of literature documents in both mice and Drosophila the involvement of Insulin pathway in growth regulation, probably due to its role in glucose and lipid import, nutrient storage, and translation of RNAs implicated in ribosome biogenesis (Vanhaesebroeck et al. 2001). Moreover several lines of evidence implicate this pathway as a causal factor in cancer (Sale, 2008; Zeng and Yee 2007; Hursting et al., 2007; Chan et al., 2008). With regards to Myc, studies in cell culture have implied this family of transcription factors as regulators of the cell cycle that are rapidly induced in response to growth factors. Myc is a potent oncogene, rearranged and overexpressed in a wide range of human tumors and necessary during development. Its conditional knock-out in mice results in reduction of body weight due to defect in cell proliferation (Trumpp et al. 2001). Evidence from in vivo studies in Drosophila and mammals suggests a critical function for myc in cell growth regulation (Iritani and Eisenman 1999; Johnston et al. 1999; Kim et al. 2000; de Alboran et al. 2001; Douglas et al. 2001). This role is supported by our analysis of Myc target genes in Drosophila, which include genes involved in RNA binding, processing, ribosome biogenesis and nucleolar function (Orain et al 2003, Bellosta et al., 2005, Hulf et al, 2005). The fact that Insulin signaling and Myc have both been associated with growth control suggests that they may interact with each other. However, genetic evidence suggesting that Insulin signaling regulates Myc in vivo is lacking. In this work we were able to show, for the first time, a direct modulation of dMyc in response to Insulin stimulation/silencing both in vitro and in vivo. Our results suggest that dMyc up-regulation in response to DILPs signaling occurs both at the mRNA and potein level. We believe dMyc protein accumulation after Insulin signaling activation is conditioned to AKT-dependent GSK3β/sgg inactivation. In fact, we were able to demonstate that dMyc protein stabilization through phosphorylation is a conserved feature between Drosophila and vertebrates and requires multiple events. The final phosphorylation step, that results in a non-stable form of dMyc protein, ready to be degraded by the proteasome, is performed by GSK3β/sgg kinase (Sears, 2004). At the same time we demonstrated that CKI family of protein kinase are required to prime dMyc phosphorylation. DILPs and TOR/Nutrient signalings are known to communicate at several levels (Neufeld, 2003). For this reason we further investigated TOR contribution to dMyc-dependent growth regulation. dMyc protein accumulates in S2 cells after aminoacid stimulation, while its mRNA does not seem to be affected upon TORC1 inhibition, suggesting that the Nutrient pathway regulates dMyc mostly post-transcriptionally. In support to this hypothesis, we observed a TORC1-dependent GSK3β/sgg inactivation, further confirming a synergic effect of DILPs and Nutrients on dMyc protein stability. On the other hand, our data show that Rheb but not S6K, both downstream of the TOR kinase, contributes to the dMyc-induced growth of the eye tissue, suggesting that Rheb controls growth independently of S6K.. Moreover, Rheb seems to be able to regulate organ size during development inducing cell death, a mechanism no longer occurring in absence of dmyc. These observations suggest that Rheb might control growth through a new pathway independent of TOR/S6K but still dependent on dMyc. In order to dissect the mechanism of dMyc regulation in response to these events, we analyzed the relative contribution of Rheb, TOR and S6K to dMyc expression, biochemically in S2 cells and in vivo in morphogenetic clones and we further confirmed an interplay between Rheb and Myc that seems to be indipendent from TOR. In this work we clarified the mechanisms that stabilize dMyc protein in vitro and in vivo and we observed for the first time dMyc responsiveness to DILPs and TOR. At the same time, we discovered a new branch of the Nutrient pathway that appears to drive growth through dMyc but indipendently from TOR. We believe our work shed light on the mechanisms cells use to grow or restrain growth in presence/absence of growth promoting cues and for this reason it contributes to understand the physiology of growth control.

Molluscs of the marine protected area "Secche di Tor Paterno"

The thesis describes the molluscan biodiversity of the infralittoral off-shore reefs in the "Secche di Tor Paterno" marine protected area lying in the Central Tyrrhenian Sea off the coasts of Lazio south of Roma. Data originate from underwater sampling activities carried out by SCUBA diving in four biocoenoses: Posidonia oceanica leaves and rhizomes, coralligenous concretions and detritic pools. The representativeness of molluscs as descriptors of biocoenoses is evaluated by preliminary comparisons with data about Polychaeta, Pleocyemata (Crustacea) and Brachiopoda obtained in the same survey. The malacocoenoses of the four biocoenoses are treated in detail. Then data are compared with other data sets to assess differences and similarities with other communities. The agreement between death and living assemblages in the reefs is evaluated for the Posidonia oceanica and the coralligenous biocoenosis and was carried out by a set of standard metrics and some benthic ecology methods. Molluscs perform very well as descriptors of biocoenoses, better than the other phyla. The molluscan assemblages of the reefs are very rich in species despite richness is mainly concentrated in the coralligenous and in the rhizomes of Posidonia oceanica. The leaves of Posidonia oceanica host a rather poor assemblage. Detritic pools host a poor but peculiar species assemblage. The dead-live agreement showed that death assemblages are highly representative of sediments of nearby biocoenoses as a result of low bottom transport. Fidelity metrics suggest a good agreement between the living and death assemblages when species richness and taxonomic composition are considered. The study suggests that fidelity is lower when considering the species dominance. These differences could be associated to the trophism of species and possibly to the species life span.

Pattern recognition analysis on heavy ion reaction data

One of the problems in the analysis of nucleus-nucleus collisions is to get information on the value of the impact parameter b. This work consists in the application of pattern recognition techniques aimed at associating values of b to groups of events. To this end, a support vec- tor machine (SVM) classifier is adopted to analyze multifragmentation reactions. This method allows to backtracing the values of b through a particular multidimensional analysis. The SVM classification con- sists of two main phase. In the first one, known as training phase, the classifier learns to discriminate the events that are generated by two different model:Classical Molecular Dynamics (CMD) and Heavy- Ion Phase-Space Exploration (HIPSE) for the reaction: 58Ni +48 Ca at 25 AMeV. To check the classification of events in the second one, known as test phase, what has been learned is tested on new events generated by the same models. These new results have been com- pared to the ones obtained through others techniques of backtracing the impact parameter. Our tests show that, following this approach, the central collisions and peripheral collisions, for the CMD events, are always better classified with respect to the classification by the others techniques of backtracing. We have finally performed the SVM classification on the experimental data measured by NUCL-EX col- laboration with CHIMERA apparatus for the previous reaction.

Competitività in sostenibilità urbana e territoriale. L’Emilia Romagna, regione d’Europa

The thesis in Urban and Regional Geography titled “URBAN AND TERRITORIAL COMPETITIVENESS IN SUSTAINABILITY. EMILIA-ROMAGNA, REGION OF EUROPE” is divided into two sections. Section one is additionally composed by two chapters (chap. 1 and 2) and deals with theoretical and gnosiological issues. Section two, of two more chapters (chap. 3 and 4), provides practical contributions: these issues give explanatory patterns to interpret the performances of emiliano-romagnoli urban systems. Chapter one is an introductory chapter. It analyzes globalization that involves a larger and larger number of cities, rich or poor. It also considers the so called “digital divide” either as one of the major phenomena of this unhomogeneous development or as an interesting gnosiological and practical challenge of geography. Globalization is now involving all the cities, large or small, but the small ones have higher risks of exclusion: it depends on their more fragile socio-economic, cultural, and environmental urban structure. That’s why European Union (chapter two) promotes policies and endows politics to sustain cities, because urban systems are the basis for the territorial development. So, European, national and local Institutions are firmly interested in promoting urban and local interventions and projects. Section two deals with economic-geography methods, which consists on collecting indicators and the benchmarking methodology. It also specifically analyzes the urban systems of Emilia-Romagna. Consequences of the globalization on the cities are interpreted with a study of their local resources, intended as potentials for their development. The STeMA approach, proposed by Professor Maria Prezioso (University of Roma, “Tor Vergata”) and used by the ESPON (European Spatial Program Observation Network) project, describes the main “determinants” of the territorial and urban development. These are easily comparable to one another (similar or better performing). This approach achieves two goals. On one hand, it is possible to analyze every urban system in its all main characteristics and to preserve its historical and cultural factors. On another hand, each city is “knowable” and “understandable” by all scholars, as it is objectively comparable. So, urban planners can propose specific “multi-level” and “multi-varied” programs of governance. These will face globalization by exalting local empowerment.

Mediterranean brachiopods: morphological, ecological and phylogenetic aspects

The Brachiopoda of the Marine Protected Area “Secche di Tor Paterno”, Central Tyrrhenian Sea, have been investigated in order to give a first glance of the diversity of the brachiopods of this area and provide a new report on the Mediterranean Brachiopod fauna. Four species were reported: Novocrania anomala (Müller, 1776), Megathiris detruncata (Gmelin, 1790), Joania cordata (Risso,1826) and Argyrotheca cuneata (Risso,1826). For all the four species a morphological analysis was carried out. For the two most abundant species, J.cordata and A. cuneata, a morphometric study, based on thickness/width and length/width scattergrams, was carried out, in order to investigate their variability. Size-frequency distributions relative to the three dimensions of the shell were also computed, aimed at a evaluation of population dynamics of these two species. The results showed that, for both species, the parameters which most determine the rise of the shell during the growth of animal are width and length and that frequency distributions are mainly bi- or plurymodal and that they are difficult to interpret, as reported by other studies. Analysis of drill holes found on the shell of some specimens of the two same species revealed a predatory origin and that three different predators are responsible for them. Partial sequences of two different genetic markers, the Internal Transcribed Spacer 1 (ITS1) and the cytochrome oxidase subunit 1 (COI), were used to investigate the phylogenetic relationship between two populations of the eurybathic brachiopod species Gryphus vitreus (Born,1778) across the strait of Gibraltar. This represents the first genetic population study on brachiopods. Results from AMOVA and Bayesian analysis performed on 31 specimens highlighted no genetic differentiation indicating a likely panmixia, dispite the lecitotrophic development of the species.

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.