Role of Notch signalling in human hepatocellular carcinoma

The Notch signalling is a cellular pathway that results conserved from Drosophila to Homo sapiens controlling a wide range of cellular processes in development and in differentiated organs. It induces cell proliferation or differentiation, increased survival or apoptosis, and it is involved in stemness maintainance. These functions are conserved, but exerted with a high tissue and cellular context specificity. Signalling activation determs nuclear translocation of the receptor’s cytoplasmic domain and activation of target genes transcription. As many developmental pathway, Notch deregulation is involved in cancer, leading to oncogenic or tumour suppressive role depending on the functions exerted in normal tissue. Notch1 and Notch3 resulted aberrantly expressed in human hepatocellular carcinoma (HCC) that is the more frequent tumour of the liver and the sixth most common tumour worldwide. This thesis has the aim to investigate the role of the signalling in HCC, with particular attention to dissect common and uncommon regulatory pathways between Notch1 and Notch3 and to define the role of the signalling in HCC. Nocth1 and Notch3 were analysed on their regulation on Hes1 target and involvement in cell cycle control. They showed to regulate CDKN1C/p57kip2 expression through Hes1 target. CDKN1C/p57kip2 induces not only cell cycle arrest, but also senescence in HCC cell lines. Moreover, the involvement of Notch1 in cancer progression and epithelial to mesenchymal transition was investigated. Notch1 showed to induce invasion of HCC, regulating EMT and E- Cadherin expression. Moreover, Notch3 showed specific regulation on p53 at post translational levels. In vitro and ex vivo analysis on HCC samples suggests a complex role of both receptors in regulate HCC, with an oncogenic role but also showing tumour suppressive effects, suggesting a complex and deep involvement of this signalling in HCC.

Mesenchymal stromal cell: new applications for regenerative medicine

In the last decades mesenchymal stromal cells (MSC), intriguing for their multilineage plasticity and their proliferation activity in vitro, have been intensively studied for innovative therapeutic applications. In the first project, a new method to expand in vitro adipose derived-MSC (ASC) while maintaining their progenitor properties have been investigated. ASC are cultured in the same flask for 28 days in order to allow cell-extracellular matrix and cell-cell interactions and to mimic in vivo niche. ASC cultured with this method (Unpass cells) were compared with ASC cultured under classic condition (Pass cells). Unpass and Pass cells were characterized in terms of clonogenicity, proliferation, stemness gene expression, differentiation in vitro and in vivo and results obtained showed that Unpass cells preserve their stemness and phenotypic properties suggesting a fundamental role of the niche in the maintenance of ASC progenitor features. Our data suggests alternative culture conditions for the expansion of ASC ex vivo which could increase the performance of ASC in regenerative applications. In vivo MSC tracking is essential in order to assess their homing and migration. Super-paramagnetic iron oxide nanoparticles (SPION) have been used to track MSC in vivo due to their biocompatibility and traceability by MRI. In the second project a new generation of magnetic nanoparticles (MNP) used to label MSC were tested. These MNP have been functionalized with hyperbranched poly(epsilon-lysine)dendrons (G3CB) in order to interact with membrane glycocalix of the cells avoiding their internalization and preventing any cytotoxic effects. In literature it is reported that labeling of MSC with SPION takes long time of incubation. In our experiments after 15min of incubation with G3CB-MNP more then 80% of MSC were labeled. The data obtained from cytotoxic, proliferation and differentiation assay showed that labeling does not affect MSC properties suggesting a potential application of G3CB nano-particles in regenerative medicine.

Adipose-derived stem cells and tissue revascularization: enhancing islet survival and performance for diabetes care.

Pancreatic islet transplantation represents a fascinating procedure that, at the moment, can be considered as alternative to standard insulin treatment or pancreas transplantation only for selected categories of patients with type 1 diabetes mellitus. Among the factors responsible for leading to poor islet engraftment, hypoxia plays an important role. Mesenchymal stem cells (MSCs) were recently used in animal models of islet transplantation not only to reduce allograft rejection, but also to promote revascularization. Currently adipose tissue represents a novel and good source of MSCs. Moreover, the capability of adipose-derived stem cells (ASCs) to improve islet graft revascularization was recently reported after hybrid transplantation in mice. Within this context, we have previously shown that hyaluronan esters of butyric and retinoic acids can significantly enhance the rescuing potential of human MSCs. Here we evaluated whether ex vivo preconditioning of human ASCs (hASCs) with a mixture of hyaluronic (HA), butyric (BU), and retinoic (RA) acids may result in optimization of graft revascularization after islet/stem cell intrahepatic cotransplantation in syngeneic diabetic rats. We demonstrated that hASCs exposed to the mixture of molecules are able to increase the secretion of vascular endothelial growth factor (VEGF), as well as the transcription of angiogenic genes, including VEGF, KDR (kinase insert domain receptor), and hepatocyte growth factor (HGF). Rats transplanted with islets cocultured with preconditioned hASCs exhibited a better glycemic control than rats transplanted with an equal volume of islets and control hASCs. Cotransplantation with preconditioned hASCs was also associated with enhanced islet revascularization in vivo, as highlighted by graft morphological analysis. The observed increase in islet graft revascularization and function suggests that our method of stem cell preconditioning may represent a novel strategy to remarkably improve the efficacy of islets-hMSCs cotransplantation.

SMO inhibitor specifically targets the Hedgehog Pathway and reverts the drug-resistance of Leukemic Stem Cells

Abnormal Hedgehog signaling is associated with human malignancies. Smo, a key player of that signaling, is the most suitable target to inhibit this pathway. To this aim several molecules, antagonists of Smo, have been synthesized, and some of them have started the phase I in clinical trials. Our hospital participated to one of these studies which investigated the oral administration of a new selective inhibitor of Smo (SMOi). To evaluate ex vivo SMOi efficacy and to identify new potential clinical biomarkers of responsiveness, we separated bone marrow CD34+ cells from 5 acute myeloid leukemia (AML), 1 myelofibrosis (MF), 2 blastic phases chronic myeloid leukemia (CML) patients treated with SMOi by immunomagnetic separation, and we analysed their gene expression profile using Affimetrix HG-U133 Plus 2.0 platform. This analysis, showed differential expression after 28 days start of therapy (p-value ≤ 0.05) of 1,197 genes in CML patients and 589 genes in AML patients. This differential expression is related to Hedgehog pathway with a p-value = 0.003 in CML patients and with a p-value = 0.0002 in AML patients, suggesting that SMOi targets specifically this pathway. Among the genes differentially expressed we observed strong up-regulation of Gas1 and Kif27 genes, which may work as biomarkers of responsiveness of SMOi treatment in CML CD34+ cells whereas Hedgehog target genes (such as Smo, Gli1, Gli2, Gli3), Bcl2 and Abca2 were down-regulated, in both AML and CML CD34+ cells. It has been reported that Bcl-2 expression could be correlated with cancer therapy resistance and that Hedgehog signaling modulate ATP-binding (ABC) cassette transporters, whose expression has been correlated with chemoresistance. Moreover we confirmed that in vitro SMOi treatment targets Hedgehog pathway, down-regulate ABC transporters, Abcg2 and Abcb1 genes, and in combination with tyrosine kinase inhibitors (TKIs) could revert the chemoresistance mechanism in K562 TKIs-resistant cell line.

Un estere misto degli acidi ialuronico, butirrico e retinoico è in grado di agire come rimodellante inverso della matrice cellulare sui fibroblasti cardiaci

Le cardiomiopatie che insorgono a seguito di infarto miocardico sono causa di elevata morbilità e mortalità dalle importanti ricadute cliniche, dovute alle patologie insorgenti a seguito dell’ischemia e della cicatrice post-infatuale. Il ventricolo sinistro danneggiato va incontro a un rimodellamento progressivo, con perdita di cardiomiociti e proliferazione dei fibroblasti, risultante in un’architettura e in una funzionalità dell’organo distorta. I fibroblasti cardiaci sono i principali responsabili della fibrosi, il processo di cicatrizzazione caratterizzato da un’eccessiva deposizione di matrice extracellulare (ECM). Negli ultimi anni gli sforzi del nostro laboratorio sono stati volti a cercare di risolvere questo problema, attraverso l’uso di una molecola da noi sintetizzata, un estere misto degli acidi butirrico, retinoico e ialuronico, HBR, capace di commissionare le cellule staminali in senso cardio-vascolare. Studi in vivo mostrano come l’iniezione diretta di HBR in cuori di animali sottoposti a infarto sperimentale, sia in grado, tra le atre cose, di diminuire la fibrosi cardiaca. Sulla base di questa evidenza abbiamo cercato di capire come e se HBR agisse direttamente sui fibroblasti, indagando i meccanismi coinvolti nella riduzione della fibrosi in vivo.. In questa tesi abbiamo dimostrato come HBR abbia un’azione diretta su fibroblasti, inibendone la proliferazione, senza effetti citotossici. Inoltre HBR induce una significativa riduzione della deposizione di collagene.. HBR agisce sull’espressione genica e sulla sintesi proteica, sopprimendo la trascrizione dei geni del collagene, così come dell’a-sma, inibendo la trasizione fibroblasti-miofibroblasti, e promuovendo la vasculogenesi (attraverso VEGF), la chemoattrazione di cellule staminali (attraverso SDF) e un’attività antifibrotica (inibendo CTGF). HBR sembra modulare l’espressione genica agendo direttamente sulle HDAC, probabilmente grazie alla subunità BU. L’abilità di HBR di ridurre la fibrosi post-infartuale, come dimostrato dai nostri studi in vivo ed in vitro, apre la strada a importanti prospettive terapeutiche.

The reconstruction of skeletal movement: the soft tissue artefact issue

In 3D human movement analysis performed using stereophotogrammetric systems and skin markers, bone pose can only be estimated in an indirect fashion. During a movement, soft tissue deformations make the markers move with respect to the underlying bone generating soft tissue artefact (STA). STA has devastating effects on bone pose estimation and its compensation remains an open question. The aim of this PhD thesis was to contribute to the solution of this crucial issue. Modelling STA using measurable trial-specific variables is a fundamental prerequisite for its removal from marker trajectories. Two STA model architectures are proposed. Initially, a thigh marker-level artefact model is presented. STA was modelled as a linear combination of joint angles involved in the movement. This model was calibrated using ex-vivo and in-vivo STA invasive measures. The considerable number of model parameters led to defining STA approximations. Three definitions were proposed to represent STA as a series of modes: individual marker displacements, marker-cluster geometrical transformations (MCGT), and skin envelope shape variations. Modes were selected using two criteria: one based on modal energy and another on the selection of modes chosen a priori. The MCGT allows to select either rigid or non-rigid STA components. It was also empirically demonstrated that only the rigid component affects joint kinematics, regardless of the non-rigid amplitude. Therefore, a model of thigh and shank STA rigid component at cluster-level was then defined. An acceptable trade-off between STA compensation effectiveness and number of parameters can be obtained, improving joint kinematics accuracy. The obtained results lead to two main potential applications: the proposed models can generate realistic STAs for simulation purposes to compare different skeletal kinematics estimators; and, more importantly, focusing only on the STA rigid component, the model attains a satisfactory STA reconstruction with less parameters, facilitating its incorporation in an pose estimator.