Study of apoptotic deletion mediated by Bifidobacterium longum with construction of recombinant strains for Serpin encoding gene and phenotypes comparison in a pig cell model

The first part of the research project of the Co-Advisorship Ph.D Thesis was aimed to select the best Bifidobacterium longum strains suitable to set the basis of our study. We were looking for strains with the abilities to colonize the intestinal mucosa and with good adhesion capacities, so that we can test these strains to investigate their ability to induce apoptosis in “damaged” intestinal cells. Adhesion and apoptosis are the two process that we want to study to better understand the role of an adhesion protein that we have previously identified and that have top scores homologies with the recent serpin encoding gene identified in B. longum by Nestlè researchers. Bifidobacterium longum is a probiotic, known for its beneficial effects to the human gut and even for its immunomodulatory and antitumor activities. Recently, many studies have stressed out the intimate relation between probiotic bacteria and the GIT mucosa and their influence on human cellular homeostasis. We focused on the apoptotic deletion of cancer cells induced by B. longum. This has been valued in vitro, performing the incubation of three B.longum strains with enterocyte-like Caco- 2 cells, to evidence DNA fragmentation, a cornerstone of apoptosis. The three strains tested were defined for their adhesion properties using adhesion and autoaggregation assays. These features are considered necessary to select a probiotic strain. The three strains named B12, B18 and B2990 resulted respectively: “strong adherent”, “adherent” and “non adherent”. Then, bacteria were incubated with Caco-2 cells to investigate apoptotic deletion. Cocultures of Caco-2 cells with B. longum resulted positive in DNA fragmentation test, only when adherent strains were used (B12 and B18). These results indicate that the interaction with adherent B. longum can induce apoptotic deletion of Caco-2 cells, suggesting a role in cellular homeostasis of the gastrointestinal tract and in restoring the ecology of damaged colon tissues. These results were used to keep on researching and the strains tested were used as recipient of recombinant techniques aimed to originate new B.longum strains with enhanced capacity of apoptotic induction in “damaged” intestinal cells. To achieve this new goal it was decided to clone the serpin encoding gene of B. longum, so that we can understand its role in adhesion and apoptosis induction. Bifidobacterium longum has immunostimulant activity that in vitro can lead to apoptotic response of Caco-2 cell line. It secretes a hypothetical eukaryotic type serpin protein, which could be involved in this kind of deletion of damaged cells. We had previously characterised a protein that has homologies with the hypothetical serpin of B. longum (DD087853). In order to create Bifidobacterium serpin transformants, a B. longum cosmid library was screened with a PCR protocol using specific primers for serpin gene. After fragment extraction, the insert named S1 was sub-cloned into pRM2, an Escherichia coli - Bifidobacterium shuttle vector, to construct pRM3. Several protocols for B. longum transformation were performed and the best efficiency was obtained using MRS medium and raffinose. Finally bacterial cell supernatants were tested in a dotblot assay to detect antigens presence against anti-antitrypsin polyclonal antibody. The best signal was produced by one starin that has been renamed B. longum BLKS 7. Our research study was aimed to generate transformants able to over express serpin encoding gene, so that we can have the tools for a further study on bacterial apoptotic induction of Caco-2 cell line. After that we have originated new trasformants the next step to do was to test transformants abilities when exposed to an intestinal cell model. In fact, this part of the project was achieved in the Department of Biochemistry of the Medical Faculty of the University of Maribor, guest of the abroad supervisor of the Co-Advisorship Doctoral Thesis: Prof. Avrelija Cencic. In this study we examined the probiotic ability of some bacterial strains using intestinal cells from a 6 years old pig. The use of intestinal mammalian cells is essential to study this symbiosis and a functional cell model mimics a polarised epithelium in which enterocytes are separated by tight junctions. In this list of strains we have included the Bifidobacterium longum BKS7 transformant strain that we have previously originated; in order to compare its abilities. B. longum B12 wild type and B. longum BKS7 transformant and eight Lactobacillus strains of different sources were co-cultured with porcine small intestine epithelial cells (PSI C1) and porcine blood monocytes (PoM2) in Transwell filter inserts. The strains, including Lb. gasseri, Lb. fermentum, Lb. reuterii, Lb. plantarum and unidentified Lactobacillus from kenyan maasai milk and tanzanian coffee, were assayed for activation of cell lines, measuring nitric oxide by Griess reaction, H202 by tetramethylbenzidine reaction and O2 - by cytochrome C reduction. Cytotoxic effect by crystal violet staining and induction on metabolic activity by MTT cell proliferation assay were tested too. Transepithelial electrical resistance (TER) of polarised PSI C1 was measured during 48 hours co-culture. TER, used to observe epithelium permeability, decrease during pathogenesis and tissue becomes permeable to ion passive flow lowering epithelial barrier function. Probiotics can prevent or restore increased permeability. Lastly, dot-blot was achieved against Interleukin-6 of treated cells supernatants. The metabolic activity of PoM2 and PSI C1 increased slightly after co-culture not affecting mitochondrial functions. No strain was cytotoxic over PSI C1 and PoM2 and no cell activation was observed, as measured by the release of NO2, H202 and O2 - by PoM2 and PSI C1. During coculture TER of polarised PSI C1 was two-fold higher comparing with constant TER (~3000 ) of untreated cells. TER raise generated by bacteria maintains a low permeability of the epithelium. During treatment Interleukin-6 was detected in cell supernatants at several time points, confirming immunostimulant activity. All results were obtained using Lactobacillus paracasei Shirota e Carnobacterium divergens as controls. In conclusion we can state that both the list of putative probiotic bacteria and our new transformant strain of B. longum are not harmful when exposed to intestinal cells and could be selected as probiotics, because can strengthen epithelial barrier function and stimulate nonspecific immunity of intestinal cells on a pig cell model. Indeed, we have found out that none of the strains tested that have good adhesion abilities presents citotoxicity to the intestinal cells and that non of the strains tested can induce cell lines to produce high level of ROS, neither NO2. Moreover we have assayed even the capacity of producing certain citokynes that are correlated with immune response. The detection of Interleukin-6 was assayed in all our samples, including B.longum transformant BKS 7 strain, this result indicates that these bacteria can induce a non specific immune response in the intestinal cells. In fact, when we assayed the presence of Interferon-gamma in cells supernatant after bacterial exposure, we have no positive signals, that means that there is no activation of a specific immune response, thus confirming that these bacteria are not recognize as pathogen by the intestinal cells and are certainly not harmful for intestinal cells. The most important result is the measure of Trans Epithelial Electric Resistance that have shown how the intestinal barrier function get strengthen when cells are exposed to bacteria, due to a reduction of the epithelium permeability. We have now a new strain of B. longum that will be used for further studies above the mechanism of apoptotic induction to “damaged cells” and above the process of “restoring ecology”. This strain will be the basis to originate new transformant strains for Serpin encoding gene that must have better performance and shall be used one day even in clinical cases as in “gene therapy” for cancer treatment and prevention.

Mitochondrial dysfunction in a cell model of thyroid oncocytoma

The role of mitochondrial dysfunction in cancer has long been a subject of great interest. In this study, such dysfunction has been examined with regards to thyroid oncocytoma, a rare form of cancer, accounting for less than 5% of all thyroid cancers. A peculiar characteristic of thyroid oncocytic cells is the presence of an abnormally large number of mitochondria in the cytoplasm. Such mitochondrial hyperplasia has also been observed in cells derived from patients suffering from mitochondrial encephalomyopathies, where mutations in the mitochondrial DNA(mtDNA) encoding the respiratory complexes result in oxidative phosphorylation dysfunction. An increase in the number of mitochondria occurs in the latter in order to compensate for the respiratory deficiency. This fact spurred the investigation into the presence of analogous mutations in thyroid oncocytic cells. In this study, the only available cell model of thyroid oncocytoma was utilised, the XTC-1 cell line, established from an oncocytic thyroid metastasis to the breast. In order to assess the energetic efficiency of these cells, they were incubated in a medium lacking glucose and supplemented instead with galactose. When subjected to such conditions, glycolysis is effectively inhibited and the cells are forced to use the mitochondria for energy production. Cell viability experiments revealed that XTC-1 cells were unable to survive in galactose medium. This was in marked contrast to the TPC-1 control cell line, a thyroid tumour cell line which does not display the oncocytic phenotype. In agreement with these findings, subsequent experiments assessing the levels of cellular ATP over incubation time in galactose medium, showed a drastic and continual decrease in ATP levels only in the XTC-1 cell line. Furthermore, experiments on digitonin-permeabilised cells revealed that the respiratory dysfunction in the latter was due to a defect in complex I of the respiratory chain. Subsequent experiments using cybrids demonstrated that this defect could be attributed to the mitochondrially-encoded subunits of complex I as opposed to the nuclearencoded subunits. Confirmation came with mtDNA sequencing, which detected the presence of a novel mutation in the ND1 subunit of complex I. In addition, a mutation in the cytochrome b subunit of complex III of the respiratory chain was detected. The fact that XTC-1 cells are unable to survive when incubated in galactose medium is consistent with the fact that many cancers are largely dependent on glycolysis for energy production. Indeed, numerous studies have shown that glycolytic inhibitors are able to induce apoptosis in various cancer cell lines. Subsequent experiments were therefore performed in order to identify the mode of XTC-1 cell death when subjected to the metabolic stress imposed by the forced use of the mitochondria for energy production. Cell shrinkage and mitochondrial fragmentation were observed in the dying cells, which would indicate an apoptotic type of cell death. Analysis of additional parameters however revealed a lack of both DNA fragmentation and caspase activation, thus excluding a classical apoptotic type of cell death. Interestingly, cleavage of the actin component of the cytoskeleton was observed, implicating the action of proteases in this mode of cell demise. However, experiments employing protease inhibitors failed to identify the specific protease involved. It has been reported in the literature that overexpression of Bcl-2 is able to rescue cells presenting a respiratory deficiency. As the XTC-1 cell line is not only respiration-deficient but also exhibits a marked decrease in Bcl-2 expression, it is a perfect model with which to study the relationship between Bcl-2 and oxidative phosphorylation in respiratory-deficient cells. Contrary to the reported literature studies on various cell lines harbouring defects in the respiratory chain, Bcl-2 overexpression was not shown to increase cell survival or rescue the energetic dysfunction in XTC-1 cells. Interestingly however, it had a noticeable impact on cell adhesion and morphology. Whereas XTC-1 cells shrank and detached from the growth surface under conditions of metabolic stress, Bcl-2-overexpressing XTC-1 cells appeared much healthier and were up to 45% more adherent. The target of Bcl-2 in this setting appeared to be the actin cytoskeleton, as the cleavage observed in XTC-1 cells expressing only endogenous levels of Bcl-2, was inhibited in Bcl-2-overexpressing cells. Thus, although unable to rescue XTC-1 cells in terms of cell viability, Bcl-2 is somehow able to stabilise the cytoskeleton, resulting in modifications in cell morphology and adhesion. The mitochondrial respiratory deficiency observed in cancer cells is thought not only to cause an increased dependency on glycolysis but it is also thought to blunt cellular responses to anticancer agents. The effects of several therapeutic agents were thus assessed for their death-inducing ability in XTC-1 cells. Cell viability experiments clearly showed that the cells were more resistant to stimuli which generate reactive oxygen species (tert-butylhydroperoxide) and to mitochondrial calcium-mediated apoptotic stimuli (C6-ceramide), as opposed to stimuli inflicting DNA damage (cisplatin) and damage to protein kinases(staurosporine). Various studies in the literature have reported that the peroxisome proliferator-activated receptor-coactivator 1(PGC-1α), which plays a fundamental role in mitochondrial biogenesis, is also involved in protecting cells against apoptosis caused by the former two types of stimuli. In accordance with these observations, real-time PCR experiments showed that XTC-1 cells express higher mRNA levels of this coactivator than do the control cells, implicating its importance in drug resistance. In conclusion, this study has revealed that XTC-1 cells, like many cancer cell lines, are characterised by a reduced energetic efficiency due to mitochondrial dysfunction. Said dysfunction has been attributed to mutations in respiratory genes encoded by the mitochondrial genome. Although the mechanism of cell demise in conditions of metabolic stress is unclear, the potential of targeting thyroid oncocytic cancers using glycolytic inhibitors has been illustrated. In addition, the discovery of mtDNA mutations in XTC-1 cells has enabled the use of this cell line as a model with which to study the relationship between Bcl-2 overexpression and oxidative phosphorylation in cells harbouring mtDNA mutations and also to investigate the significance of such mutations in establishing resistance to apoptotic stimuli.

Molecular bases, pathogenic mechanisms and possible therapeutic approach in Leber's Hereditary Optic Neuropathy

Leber’s hereditary optic neuropathy (LHON) is a mitochondrial disease characterized by a rapid loss of central vision and optic atrophy, due to the selective degeneration of retinal ganglion cells. The age of onset is around 20, and the degenerative process is fast and usually the second eye becomes affected in weeks or months. Even if this pathology is well known and has been well characterized, there are still open questions on its pathophysiology, such as the male prevalence, the incomplete penetrance and the tissue selectivity. This maternally inherited disease is caused by mutations in mitochondrial encoded genes of NADH ubiquinone oxidoreductase (complex I) of the respiratory chain. The 90% of LHON cases are caused by one of the three common mitochondrial DNA mutations (11778/ND4, 14484/ND6 and 3460/ND1) and the remaining 10% is caused by rare pathogenic mutations, reported in literature in one or few families. Moreover, there is also a small subset of patients reported with new putative pathogenic nucleotide changes, which awaits to be confirmed. We here clarify some molecular aspects of LHON, mainly the incomplete penetrance and the role of rare mtDNA mutations or variants on LHON expression, and attempt a possible therapeutic approach using the cybrids cell model. We generated novel structural models for mitochondrial encoded complex I subunits and a conservation analysis and pathogenicity prediction have been carried out for LHON reported mutations. This in-silico approach allowed us to locate LHON pathogenic mutations in defined and conserved protein domains and can be a useful tool in the analysis of novel mtDNA variants with unclear pathogenic/functional role. Four rare LHON pathogenic mutations have been identified, confirming that the ND1 and ND6 genes are mutational hot spots for LHON. All mutations were previously described at least once and we validated their pathogenic role, suggesting the need for their screening in LHON diagnostic protocols. Two novel mtDNA variants with a possible pathogenic role have been also identified in two independent branches of a large pedigree. Functional studies are necessary to define their contribution to LHON in this family. It also been demonstrated that the combination of mtDNA rare polymorphic variants is relevant in determining the maternal recurrence of myoclonus in unrelated LHON pedigrees. Thus, we suggest that particular mtDNA backgrounds and /or the presence of specific rare mutations may increase the pathogenic potential of the primary LHON mutations, thereby giving rise to the extraocular clinical features characteristic of the LHON “plus” phenotype. We identified the first molecular parameter that clearly discriminates LHON affected individuals from asymptomatic carriers, the mtDNA copy number. This provides a valuable mechanism for future investigations on variable penetrance in LHON. However, the increased mtDNA content in LHON individuals was not correlated to the functional polymorphism G1444A of PGC-1 alpha, the master regulator of mitochondrial biogenesis, but may be due to gene expression of genes involved in this signaling pathway, such as PGC-1 alpha/beta and Tfam. Future studies will be necessary to identify the biochemical effects of rare pathogenic mutations and to validate the novel candidate mutations here described, in terms of cellular bioenergetic characterization of these variants. Moreover, we were not able to induce mitochondrial biogenesis in cybrids cell lines using bezafibrate. However, other cell line models are available, such as fibroblasts harboring LHON mutations, or other approaches can be used to trigger the mitochondrial biogenesis.

Respiratory chain complex I dysfunction in tumorigenesis

Diseases due to mutations in mitochondrial DNA probably represent the most common form of metabolic disorders, including cancer, as highlighted in the last years. Approximately 300 mtDNA alterations have been identified as the genetic cause of mitochondrial diseases and one-third of these alterations are located in the coding genes for OXPHOS proteins. Despite progress in identification of their molecular mechanisms, little has been done with regard to the therapy. Recently, a particular gene therapy approach, namely allotopic expression, has been proposed and optimized, although the results obtained are rather controversial. In fact, this approach consists in synthesis of a wild-type version of mutated OXPHOS protein in the cytosolic compartment and in its import into mitochondria, but the available evidence is based only on the partial phenotype rescue and not on the demonstration of effective incorporation of the functional protein into respiratory complexes. In the present study, we took advantage of a previously analyzed cell model bearing the m.3571insC mutation in MTND1 gene for the ND1 subunit of respiratory chain complex I. This frame-shift mutation induces in fact translation of a truncated ND1 protein then degraded, causing complex I disassembly, and for this reason not in competition with that allotopically expressed. We show here that allotopic ND1 protein is correctly imported into mitochondria and incorporated in complex I, promoting its proper assembly and rescue of its function. This result allowed us to further confirm what we have previously demonstrated about the role of complex I in tumorigenesis process. Injection of the allotopic clone in nude mice showed indeed that the rescue of complex I assembly and function increases tumor growth, inducing stabilization of HIF1α, the master regulator of tumoral progression, and consequently its downstream gene expression activation.

Ricerca di nuove strategie differenziative: Analisi degli effetti di stimoli fisici e molecolari su cellule mesenchimali umane isolate da tessuto adiposo

Introduzione. Le cellule mesenchimali derivate dal tessuto adiposo (hASC) rappresentano un importante strumento per la terapia cellulare, in quanto derivano da un tessuto adulto abbondante e facilmente reperibile. Con il dispositivo medico Lipogems l’isolamento di tali cellule è eseguito esclusivamente mediante sollecitazioni meccaniche. Il prodotto ottenuto è quindi minimamente manipolato e subito utilizzabile. Ad oggi, il condizionamento pro-differenziativo delle staminali è per lo più attuato mediante molecole di sintesi. Tuttavia, altri fattori possono modulare la fisiologia cellulare, come gli stimoli fisici e molecole naturali. Onde elettromagnetiche hanno indotto in modelli cellulari staminali l’espressione di alcuni marcatori di differenziamento e, in cellule adulte, una riprogrammazione, mentre estratti embrionali di Zebrafish sono risultati antiproliferativi sia in vitro che in vivo. Metodi. La ricerca di nuove strategie differenziative sia di natura fisica che molecolare, nel particolare onde acustiche ed estratti embrionali di Zebrafish, è stata condotta utilizzando come modello cellulare le hASC isolate con Lipogems. Onde acustiche sono state somministrate mediante l’utilizzo di due apparati di trasduzione, un generatore di onde meccaniche e il Cell Exciter . I trattamenti con gli estratti embrionali sono stati effettuati utilizzando diverse concentrazioni e diversi tempi sperimentali. Gli effetti sull’espressione dei marcatori di staminalità e differenziamento relativi ai trattamenti sono stati saggiati in RT-PCR quantitativa relativa e/o in qPCR. Per i trattamenti di tipo molecolare è stata valutata anche la proliferazione. Risultati e conclusioni. La meta-analisi dei dati delle colture di controllo mostra la stabilità d’espressione genica del modello. I trattamenti con i suoni inducono variazioni dell’espressione genica, suggerendo un ruolo regolatorio di tali stimoli, in particolare del processo di commitment cardiovascolare. Due degli estratti embrionali di Zebrafish testati inibiscono la proliferazione alle 72 ore dalla somministrazione. L’analisi d’espressione associata ai trattamenti antiproliferativi suggerisce che tale effetto abbia basi molecolari simili ai processi di differenziamento.

Computational insight into materials properties

The aim of the work was to explore the practical applicability of molecular dynamics at different length and time scales. From nanoparticles system over colloids and polymers to biological systems like membranes and finally living cells, a broad range of materials was considered from a theoretical standpoint. In this dissertation five chemistry-related problem are addressed by means of theoretical and computational methods. The main results can be outlined as follows. (1) A systematic study of the effect of the concentration, chain length, and charge of surfactants on fullerene aggregation is presented. The long-discussed problem of the location of C60 in micelles was addressed and fullerenes were found in the hydrophobic region of the micelles. (2) The interactions between graphene sheet of increasing size and phospholipid membrane are quantitatively investigated. (3) A model was proposed to study structure, stability, and dynamics of MoS2, a material well-known for its tribological properties. The telescopic movement of nested nanotubes and the sliding of MoS2 layers is simulated. (4) A mathematical model to gain understaning of the coupled diffusion-swelling process in poly(lactic-co-glycolic acid), PLGA, was proposed. (5) A soft matter cell model is developed to explore the interaction of living cell with artificial surfaces. The effect of the surface properties on the adhesion dynamics of cells are discussed.

OPA1 isoforms and protein domains in the rescue of mitochondrial dysfunctions

Mutations in OPA1 gene have been identified in the majority of patients with Dominant Optic Atrophy (DOA), a blinding disease, and the syndromic form DOA-plus. OPA1 protein is a mitochondrial GTPase involved in various mitochondrial functions, present in humans in eight isoforms, resulting from alternative splicing and proteolytic processing. In this study we have investigated the specific role of each isoform through expression in OPA-/- MEFs, by evaluating their ability to improve the defective mitochondrial phenotypes. All isoforms were able to rescue the energetic efficiency, mitochondrial DNA (mtDNA) content and cristae integrity, but only the presence of both long and short forms could recover the mitochondrial morphology. In order to identify the OPA1 protein domains crucial for its functions, we selected and modified the isoform 1, shown to be one of the most efficient in preserving mitochondrial phenotype, to express three specific OPA1 variants, namely: one with a different N-terminus portion, one unable to generate short form owing to deletion of S1 cleavage site and one with a defective GTPase domain. We demonstrated that the simultaneous presence of the N- and C-terminus of OPA1 was essential for the mtDNA maintenance; a cleavable isoform generating s-forms was necessary to completely rescue the energetic competence and the presence of the C-terminus was sufficient to partially recover the cristae ultrastructure. Lastly, several pathogenic OPA1 mutations were inserted in MEF clones and the biochemical features investigated, to correlate the defective phenotypes with the clinical severity of patients. Our results clearly indicate that this cell model reflects very well the clinical characteristics of the patients, and therefore can be proposed as an useful tool to shed light on the pathomechanism underlying DOA.

Ruolo del gene umano CYYR1: dallo studio funzionale dell'ortologo in Danio rerio alla potenziale implicazione in processi tumorigenici

Il locus CYYR1 identificato e clonato sul cromosoma 21 umano è stato caratterizzato dal punto di vista molecolare come un sistema multitrascritto, esclusivo dei vertebrati che ad oggi è orfano di una funzione specifica. Dati presenti in lettura e rintracciati mostrano una possibile relazione tra il gene CYYR1 e il pathway di Sonic Hedgehog (SHH). In questo progetto di tesi è stato utilizzato il modello animale Danio rerio per indagare il ruolo funzionale dell’ortologo (cyyr1), attraverso esperimenti di gain e loss of function che hanno permesso di dimostrare un suo coinvolgimento nello sviluppo del sistema nervoso centrale, del cuore e del tessuto muscolare. Lo studio dell’ortologo in zebrafish è stato associato all’utilizzo di linee cellulari di rabdomiosarcoma umano. I risultati ottenuti dall’induzione al differenziamento miogenico di queste linee, insieme ai dati ottenuti in Danio rerio, confermano il possibile coinvolgimento del gene CYYR1 nella miogenesi. Lo studio delle relazione tra il pathway di SHH e l’espressione del gene CYYR1 è stato condotto in entrambi i modelli con l’utilizzo di differenti inibitori della via di segnalazione. I risultati ottenuti mostrano che sistemi inibitori agenti direttamente sul recettore SMO riducono l’espressione del gene. Un dato inaspettato in Danio rerio ottenuto durante questi esperimenti di inibizione, ha aperto una nuova linea di ricerca in collaborazione con l’Università di Warwick tesa a verificare la relazione tra il gene cyyr1 e il gene lefty1. Gli esperimenti condotti presso il laboratorio della Prof.ssa Sampath hanno dimostrato la localizzazione del prodotto proteico cyyr1 in Danio rerio e indagato co-localizzazioni con la proteina lefty1. Infine, in collaborazione con Dr. Deflorian e della Prof.ssa Pistocchi, è stato generato un mutante di Danio rerio deleto per il gene cyyr1 con la tecnica CRISPR/Cas9. La caratterizzazione del mutante cyyr1 -/- ha confermato alcuni dei dati ottenuti attraverso esperimenti di loss of function.

Comprehensive characterization of SDH-deficient GIST using NGS data and iPSC models

Gastrointestinal stromal tumors (GIST) are the most common di tumors of the gastrointestinal tract, arising from the interstitial cells of Cajal (ICCs) or their precursors. The vast majority of GISTs (75–85% of GIST) harbor KIT or PDGFRA mutations. A small percentage of GIST (about 10‐15%) do not harbor any of these driver mutations and have historically been called wild-type (WT). Among them, from 20% to 40% show loss of function of the succinate dehydrogenase complex (SDH), also defined as SDH‐deficient GIST. SDH-deficient GISTs display distinctive clinical and pathological features, and can be sporadic or associated with Carney triad or Carney-Stratakis syndrome. These tumors arise most frequently in the stomach with predilection to distal stomach and antrum, have a multi-nodular growth, display a histological epithelioid phenotype, and present frequent lympho-vascular invasion. Occurrence of lymph node metastases and indolent course are representative features of SDH-deficient GISTs. This subset of GIST is known for the immunohistochemical loss of succinate dehydrogenase subunit B (SDHB), which signals the loss of function of the entire SDH-complex. The overall aim of my PhD project consists of the comprehensive characterization of SDH deficient GIST. Throughout the project, clinical, molecular and cellular characterizations were performed using next-generation sequencing technologies (NGS), that has the potential to allow the identification of molecular patterns useful for the diagnosis and development of novel treatments. Moreover, while there are many different cell lines and preclinical models of KIT/PDGFRA mutant GIST, no reliable cell model of SDH-deficient GIST has currently been developed, which could be used for studies on tumor evolution and in vitro assessments of drug response. Therefore, another aim of this project was to develop a pre-clinical model of SDH deficient GIST using the novel technology of induced pluripotent stem cells (iPSC).

Kinetic and mechanistic studies on peroxidation and stabilization of lipids with industrial and biological relevance

Lipid peroxidation is a complex mechanism that causes the degradation of lipid material of both industrial and biological significance. During processing, it is known that thermal stress produces oxidation and polymerization of oils. Additionally, biological lipids with both structural and bioactive roles are prone to peroxidation, which can have pathogenic effects including cancer and long-term degenerative disorders. To create innovative strategies to slow down the deterioration of lipids, it is crucial to improve our understanding of oxidation reactions and kinetics. To this purpose, Chapter II of this thesis focuses on the kinetic study of the oxidation reactions that take place during the thermal processing of bio-oils for industrial application. Through a new method it was possible to evaluate the kinetic parameters of oxidation of various lipid materials. This allowed us to distinguish between the different lipid materials based on their intrinsic properties. The effect of 18 antioxidants from the major families of natural and synthetic phenols were studied using the same methodology in order to acquire crucial data for enhancing the antioxidant activity of phenols based on structure-activity at high temperatures. Finally, it has been described how the antioxidant activity of α-tocopherol, revealed to be scarce in our conditions, can be improved in the presence of gamma-terpinene, through a synergistic action. Chapter III describes the synthesis and study of the antioxidant activity of polydopamine nanoparticles, in order to clarify the unclear mechanism of action of this material. Finally, in Chapter IV it was reported how the gamma-terpinene strongly inhibits the peroxidation of unsaturated lipids in heterogeneous model systems (micelles and liposomes) by forming hydroperoxyl radicals which diffuse outside the lipid nucleus, blocking the propagation of the chain radical. Furthermore, gamma-terpinene shows a very potent protective activity against ferroptosis being effective in the nanomolar range in the human neuroblastoma cell model.

A SMO inhibitor drug reduces the progenitor cell's maintenance in the Drosophila hematopoietic organ: a novel model to study Chronic Myelogenous Leukemia relapse

The chronic myeloid leukemia complexity and the difficulties of disease eradication have recently led to the development of drugs which, together with the inhibitors of TK, could eliminate leukemia stem cells preventing the occurrence of relapses in patients undergoing transplantation. The Hedgehog (Hh) signaling pathway positively regulates the self-renewal and the maintenance of leukemic stem cells and not, and this function is evolutionarily conserved. Using Drosophila as a model, we studied the efficacy of the SMO inhibitor drug that inhibit the human protein Smoothened (SMO). SMO is a crucial component in the signal transduction of Hh and its blockade in mammals leads to a reduction in the disease induction. Here we show that administration of the SMO inhibitor to animals has a specific effect directed against the Drosophila ortholog protein, causing loss of quiescence and hematopoietic precursors mobilization. The SMO inhibitor induces in L3 larvae the appearance of melanotic nodules generated as response by Drosophila immune system to the increase of its hemocytes. The same phenotype is induced even by the dsRNA:SMO specific expression in hematopoietic precursors of the lymph gland. The drug action is also confirmed at cellular level. The study of molecular markers has allowed us to demonstrate that SMO inhibitor leads to a reduction of the quiescent precursors and to an increase of the differentiated cells. Moreover administering the inhibitor to heterozygous for a null allele of Smo, we observe a significant increase in the phenotype penetrance compared to administration to wild type animals. This helps to confirm the specific effect of the drug itself. These data taken together indicate that the study of inhibitors of Smo in Drosophila can represent a useful way to dissect their action mechanism at the molecular-genetic level in order to collect information applicable to the studies of the disease in humans.

Study of Bone Biomineralization in 2D and 3D Cultures of a Human Osteosarcoma Cell Line As Osteoblast-like Model

This PhD project focuses on the study of the early stages of bone biomineralization in 2D and 3D cultures of osteoblast-like SaOS-2 osteosarcoma cells, exposed to an osteogenic cocktail. The efficacy of osteogenic treatment was assessed on 2D cell cultures after 7 days. A large calcium minerals production, an overexpression of osteogenic markers and of alkaline phosphatase activity occurred in treated samples. TEM microscopy and cryo-XANES micro-spectroscopy were performed for localizing and characterizing Ca-depositions. These techniques revealed a different localization and chemical composition of Ca-minerals over time and after treatment. Nevertheless, the Mito stress test showed in treated samples a significant increase in maximal respiration levels associated to an upregulation of mitochondrial biogenesis indicative of an ongoing differentiation process. The 3D cell cultures were realized using two different hydrogels: a commercial collagen type I and a mixture of agarose and lactose-modified chitosan (CTL). Both biomaterials showed good biocompatibility with SaOS-2 cells. The gene expression analysis of SaOS-2 cells on collagen scaffolds indicated an osteogenic commitment after treatment. and Alizarin red staining highlighted the presence of Ca-spots in the differentiated samples. In addition, the intracellular magnesium quantification, and the X-ray microscopy on mineral depositions, suggested the incorporation of Mg during the early stages of bone formation process., SaOS-2 cells treated with osteogenic cocktail produced Ca mineral deposits also on CTL/agarose scaffolds, as confirmed by alizarin red staining. Further studies are underway to evaluate the differentiation also at the genetic level. Thanks to the combination of conventional laboratory methods and synchrotron-based techniques, it has been demonstrated that SaOS-2 is a suitable model for the study of biomineralization in vitro. These results have contributed to a deeper knowledge of biomineralization process in osteosarcoma cells and could provide new evidences about a therapeutic strategy acting on the reversibility of tumorigenicity by osteogenic induction.

Cancers of unknown primary site (CUPs) as a model of metastatic process.

Cancers of unknown primary site (CUPs) are a rare group of metastatic tumours, with a frequency of 3-5%, with an overall survival of 6-10 month. The identification of tumour primary site is usually reached by a combination of diagnostic investigations and immunohistochemical testing of the tumour tissue. In CUP patients, these investigations are inconclusive. Since international guidelines for treatment are based on primary site indication, CUP treatment requires a blind approach. As a consequence, CUPs are usually empiric treated with poorly effective. In this study, we applied a set of microRNAs using EvaGreen-based Droplet Digital PCR in a retrospective and prospective collection of formalin-fixed paraffin-embedded tissue samples. We assessed miRNA expression of 155 samples including primary tumours (N=94), metastases of known origin (N=10) and metastases of unknown origin (N=50). Then, we applied the shrunken centroids predictive algorithm to obtain the CUP’s site(s)-of-origin. The molecular test was successfully applied to all CUP samples and provided a site-of-origin identification for all samples, potentially within a one-week time frame from sample inclusion. In the second part of the study we derived two CUP cell lines, and corresponding patient-derived xenografts (PDXs). CUP cell lines and PDXs underwent histological, molecular, and genomic characterization confirming the features of the original tumour. Tissues-of-origin prediction was obtained from the tumour microRNA expression profile and confirmed by single cell RNA sequencing. Genomic testing analysis identified FGFR2 amplification in both models. Drug-screening assays were performed to test the activity of FGFR2-targeting drug and the combination treatment with the MEK inhibitor trametinib, which proved to be synergic and exceptionally active, both in vitro and in vivo. In conclusion, our study demonstrated that miRNA expression profiling could be employed as diagnostic test. Then we successfully derived two CUP models from patients, used for therapy tests, bringing personalized therapy closer to CUP patients.

Developing a radiomic based machine learning model to identify patients with resected non-small-cell lung cancer at high risk of relapse

Background There is a wide variation of recurrence risk of Non-small-cell lung cancer (NSCLC) within the same Tumor Node Metastasis (TNM) stage, suggesting that other parameters are involved in determining this probability. Radiomics allows extraction of quantitative information from images that can be used for clinical purposes. The primary objective of this study is to develop a radiomic prognostic model that predicts a 3 year disease free-survival (DFS) of resected Early Stage (ES) NSCLC patients. Material and Methods 56 pre-surgery non contrast Computed Tomography (CT) scans were retrieved from the PACS of our institution and anonymized. Then they were automatically segmented with an open access deep learning pipeline and reviewed by an experienced radiologist to obtain 3D masks of the NSCLC. Images and masks underwent to resampling normalization and discretization. From the masks hundreds Radiomic Features (RF) were extracted using Py-Radiomics. Hence, RF were reduced to select the most representative features. The remaining RF were used in combination with Clinical parameters to build a DFS prediction model using Leave-one-out cross-validation (LOOCV) with Random Forest. Results and Conclusion A poor agreement between the radiologist and the automatic segmentation algorithm (DICE score of 0.37) was found. Therefore, another experienced radiologist manually segmented the lesions and only stable and reproducible RF were kept. 50 RF demonstrated a high correlation with the DFS but only one was confirmed when clinicopathological covariates were added: Busyness a Neighbouring Gray Tone Difference Matrix (HR 9.610). 16 clinical variables (which comprised TNM) were used to build the LOOCV model demonstrating a higher Area Under the Curve (AUC) when RF were included in the analysis (0.67 vs 0.60) but the difference was not statistically significant (p=0,5147).