Inhibition of polarity pathways as a synthetic lethal approach to aid targeted therapy in preventing breast tumor relapse.

Breast cancer is the second most common cancer worldwide, and the one of the leading causes of cancer death in women. Due to recent advances in therapeutic approaches and early detection programs the breast cancer survival rate has seen a dramatic improvement. Consequently, it is now breast cancer recurrence, not the primary tumor, which is the leading cause of breast cancer related death. These relapses are due to malignant cells that survive treatment. Therefore, further characterization of the treatment-resistant cells is needed in order to improve the therapeutic approaches. The overexpression of oncogenes drives tumorigenesis. In human breast cancer the epidermal growth factor receptor type-2 (Her2 also called Neu) is overexpressed in 20% of breast cancer, whereas the oncogenic transcription factor c-Myc is over-expressed in 16% of breast cancers. We model breast cancer through the use of an inducible mouse model in which the oncogenes NEU and c-MYC are under the control of a doxycycline inducible promoter. By administering a diet supplemented with doxycycline we are able to induce tumorigenesis, follow primary tumor development, and additionally inactivate the oncogene expression through withdrawal of doxycycline from the mouse diet. In essence, this would mimic a perfect therapeutic setting in which we are able to completely eradicate expression of the oncogenes, yet, these mice eventually succumb to tumor recurrence in the majority of cases. Therefore the aim of my thesis is to eliminate or disrupt the treatment-resistant cells so that they are unable to give rise to the tumor relapses even after the oncogene withdrawal. We have taken an in vitro approach to screen candidates, which would interfere with the re-formation of the mammary gland following oncogene inactivation. To do so, we cultured primary mouse mammary epithelial cells, taken from the transgenic mice, in an in vitro 3D organotypic culture system. The structures, which grow in vitro closely, resemble the polarized, lumen filled acini which mammary glands are comprised of. In these cultures, we tested different inhibitors for molecules which we confirmed were activated within the tumors derived from the c-MYC/NEU mouse model. Specifically, we tested inhibitors for Focal Adhesion Kinase, Integrin Linked Kinase, for signaling pathways, which regulate survival and membrane polarity as well an inhibitory antibody for E-cadherin bridges, which play an important role in establishing cell to cell contacts and cellular epithelial polarity. Initial results of the in vitro assay show a decrease in the number of cells surviving following treatment with inhibitors. In order to determine the characteristics of the cells surviving treatment and their tumorigenic potential, we injected them into the cleared mammary fat pad of immunodeficient mice. In the meantime using inducing a second round of induction in vitro we have seen that the survivors show reduced ability to re-induce. We also performed immunofluorescence staining on the treated cells to investigate the effect of the inhibitors on the re-polarized acinar structures. The first indications from these experiments hint that introduction of inhibitors at the time of oncogene inactivation severely disrupts reconstitution of the polarized acinar mammary gland structure. A clear distribution, ie (basal/luminal) of the structural molecular components, ZO-1, E-cad was missing, suggesting an inhibition in the re-establishment of polarity in the surviving structures. Further studies are necessary to conclude that these inhibitors are able to target the treatment-resistant cells, but our first promising results encourage this line of research

Pisa. Le chiese scomparse nel terziere di San Francesco.

Passeggiando per Pisa ancora oggi ci si stupisce della quantità delle chiese esistenti nel centro storico. In realtà esse non costituiscono che una piccola parte degli edifici religiosi che costellavano la città medievale e moderna, il cui sviluppo urbanistico si dimostrò fortemente condizionato dalla fitta concentrazione in aree ristrette di chiese ed oratori, e inevitabilmente mutato in seguito al loro diradamento. Il presente studio si collega alla ricerca svolta da Chiara Zucchellini nella tesi di laurea intitolata Pisa. Le chiese scomparse nel terziere di Chinzica, che delinea la storia di otto chiese parrocchiali, esistenti nel Seicento, ma oggi scomparse, situate nella porzione meridionale della città. Le chiese studiate nella presente ricerca si trovavano invece nel settore cittadino a nord dell'Arno e in particolare nella porzione collocata ad est delle mura altomedievali, comprendente l'intero terziere di San Francesco e parte di quello di Santa Maria. La scelta degli edifici religiosi non è dunque motivata, come per il caso di Chinzica, dal ruolo di parrocchiale svolto, ma semplicemente dalla loro posizione nell'area citata. Si tratta di otto chiese non più esistenti, ma contenute nella Descrizione delle chiese, monasteri e oratori della città di Pisa intra moenia, manoscritto redatto dal canonico Paolo Tronci alla metà del XVII secolo: San Lorenzo alla Rivolta, San Simone al Parlascio, San Iacopo in Mercato, San Barnaba, San Luca Evangelista, San Francesco Compagnia, San Girolamo e Santa Elisabetta. L'obiettivo è ripercorrere la loro storia a partire dalla metà del Seicento fino alla soppressione e demolizione o accorpamento in edifici successivi, sottolineandone il legame con lo sviluppo urbanistico della zona. Tutte le chiese prese in esame scomparvero tra la fine XVIII e l'inizio del XIX secolo, in linea con le politiche di soppressione adottate proprio in questo periodo storico. Non esiste uno studio esauriente su queste chiese e la bibliografia a riguardo è limitata al solo periodo medievale e spesso esclusivamente alla loro fondazione. Di alcune si hanno notizie accessorie derivanti dai grossi sventramenti ottocenteschi subiti dall'area esaminata, di altre rimangono in loco emergenze inconfutabili della loro presenza, in altri casi testimoniata dai toponimi oramai radicati nel tempo, ma che appaiono oggi privi di un riferimento plausibile agli occhi del passante: dietro molte vie e piazze dedicate a un Santo si nasconde spesso l'esistenza di un edificio religioso ad esso dedicato, ma ormai scomparso

Resolving the Structural Debate for the Hydrated Excess Proton in Water

It has long been proposed that the hydrated excess proton in water (aka the solvated "hydronium" cation) likely has two limiting forms, that of the Eigen cation (H9O4+) and that of the Zundel cation (H5O2+). There has been debate over which of these two is the more dominant species and/or whether intermediate (or "distorted") structures between these two limits are the more realistic representation. Spectroscopy experiments have recently provided further results regarding the excess proton. These experiments show that the hydrated proton has an anisotropy reorientation time scale on the order of 1-2 ps. This time scale has been suggested to possibly contradict the picture of the more rapid "special pair dance" phenomenon for the hydrated excess proton, which is a signature of a distorted Eigen cation. The special pair dance was predicted from prior computational studies in which the hydrated central core hydronium structure continually switches (O–H···O)* special pair hydrogen-bond partners with the closest three water molecules, yielding on average a distorted Eigen cation with three equivalent and dynamically exchanging distortions. Through state-of-art simulations it is shown here that anisotropy reorientation time scales of the same magnitude are obtained that also include structural reorientations associated with the special pair dance, leading to a reinterpretation of the experimental results. These results and additional analyses point to a distorted and dynamic Eigen cation as the most prevalent hydrated proton species in aqueous acid solutions of dilute to moderate concentration, as opposed to a stabilized or a distorted (but not "dancing") Zundel cation

MiT family translocation renal cell carcinoma: looking for diagnostic and druggable markers

Renal cell carcinomas with chromosome translocation are rare neoplasms which often occur in young patients. In the last World Health Organization (WHO 2016) are named as MiT family translocation renal cell carcinoma comprising two different entities: Xp11 renal cell carcinoma and t(6;11) renal cell carcinoma. Recently, renal cell carcinomas with TFEB amplification has been described in connection with t(6;11) renal cell carcinoma. We analyzed 30 MiT family translocation renal cell carcinoma and 2 renal cell carcinomas with TFEB amplification collecting data on clinical, histological, immunohistochemical and molecular features. In this study, we sought 1) immunohistochemical diagnostic markers (cathepsin K, CD68 (PG-M1), PAX8) since the differential diagnosis is challenging, especially with pure epithelioid PEComa/epithelioid angiomyolipoma; 2) fluorescence in situ hybridization diagnostic features to reach the correct diagnosis; 3) predictive markers (MET, AXL, VEGF) in tumor tissue for target therapy. Histologically, either cytological or architectural appearance was peculiar in each case. By immunohistochemistry, almost all MiT family translocation renal cell carcinomas expressed PAX8. Staining for cathepsin K was found in 65% of Xp11 renal cell carcinomas, only a few cases were positive for melanogenic markers and all cases were negative for CD68 (PG-M1 clone). All t(6;11) renal cell carcinomas labelled for cathepsin K and Melan-A and negative for CD68 (PG-M1 clone). Seven pure epithelioid PEComa /epithelioid angiomyolipomas, used as control, were positive for cathepsin K, melanocytic markers and CD68 (PG-M1) and negative for PAX8. All MiT family translocation renal cell carcinomas were negative for AXL; 61% of Xp11 renal cell carcinomas and 5 of 7 t(6;11) renal cell carcinomas expressed MET. Fluorescence in situ hybridization results showed the presence of TFEB gene translocation in all t(6;11) renal cell carcinomas and TFE3 gene translocation in all Xp11 renal cell carcinoma with a high frequency of split fluorescent signals (mean 74% and 68% respectively). Among the eight t(6;11) renal cell carcinomas, one case displayed a high level of TFEB gene amplification and two showed increased TFEB gene copy number (3-4 copies of fluorescent signals) with a concomitant increased number of CEP6. Those three cases behaved aggressively. By FISH, VEGFA was amplified in all three cases with TFEB amplification and increased VEGFA gene copy number was observed in the two aggressive cases t(6;11) renal cell carcinomas with an overlapping increased number of TFEB fluorescent signals. Overall, VEGFA mRNA expression was observed in 8 of 10 cases (80%); of these 8 cases, three cases showed high level TFEB amplification, one case showed TFEB rearrangement with increased TFEB gene copy number, while four showed TFEB gene rearrangement without increased copy number. In conclusion, we report the high frequency of split signals by FISH in MiT family translocation renal cell carcinomas suggesting that 40% of split signals could be used as the proper cut-off to reach the correct diagnosis. We demonstrate the usefulness of CD68 (PG-M1) immunohistochemical staining in distinguishing MiT family translocation renal cell carcinoma from pure epithelioid PEComa/epithelioid angiomyolipoma. Finally, VEGF, MET but not AXL may be potential predictive marker for targeted therapy in MiT family renal cell carcinomas

Out-of-plane seismic response of masonry façades using discrete macro-element and rigid block models

This paper investigates the out-of-plane response of masonry façades under earthquakes by means of two different approaches. A discrete macro-element approach, based on modelling the structure by means of spatial deformable macro-elements interacting through nonlinear zero-thickness interfaces, and the classical approach in which the masonry façade is assumed as a rigid block subjected to earthquake loading. The latter method neglects the elasticity of the masonry element and contemplates the energy dissipation only at each impact by means of a coefficient of restitution. The results of dynamic non-linear analyses, performed with the two methods on a real case of a church façade, provide a first comparison between the two ap-proaches highlighting some limits of application of the simplified rigid block model

3D macroelement approach for nonlinear FE analysis of URM components subjected to in-plane and out-of-plane cyclic loading

The paper presents a novel 3D macroelement approach for efficient and accurate nonlinear analysis of unreinforced masonry components subjected to in-plane and out-of-plane cyclic loading. A macroscopic description for masonry is employed, where macroelements, consisting of deformable blocks interacting through cohesive interfaces, are used to represent large portions of masonry walls, enhancing computational efficiency. Enriched kinematic characteristics are adopted for the homogeneous blocks, where in-plane shear and out-of-plane bending modes are described by two independent Lagrangian parameters. Moreover, a detailed material model for the nonlinear interfaces connecting adjacent elements enables an accurate representation of complex failure modes and cracking patterns in masonry walls. As a result, the proposed FE strategy can be employed for accurate response predictions of large masonry structures subjected to cyclic loading conditions. The accuracy of the macroelement approach is validated through comparisons against results of experimental tests of solid and perforated masonry walls under in-plane and out-of-plane loading

A procedure for the identification of multiple cracks on beams and frames by static measurements

In this work, a model of the Euler-Bernoulli beam in presence of multiple-concentrated open cracks, based on the adoption of a localized flexibility model, is adopted. The closed-form solution in terms of transversal displacements due to static loads and general boundary condition is exploited to propose an inverse damage identification procedure. The proposed identification procedure does not require any solution algorithm, on the contrary is formulated by means of simple explicit sequential expressions for the crack positions and intensities including the identification of the integration constants. The number of possible detected cracks depends on the couples of adopted sensors. Undamaged beam zones can also be easily detected in relation to the sensor positions. The analytical character of the explicit expressions of the identification procedure makes the inverse formulation applicable to damaged beams included in more complex frame structures. The proposed procedure is applied for the identification of the number, position, and intensity of the cracks along simple straight beams and also to more complex frame structures with the aim of showing its simplicity for engineering applications. In addition, the robustness of the methodology here described is shown through an accurate analysis of the basic assumptions on which the theory relies and by means of a study of the effect of noise on the identification results