Thèses sur le sujet « BIOLOGIA APPLICATA MED »

Gli ormoni steroidei sono una classe di composti che regolano specifiche funzioni endocrine o non-endocrine dell'organismo quali, per esempio, i processi riproduttivi, il comportamento sessuale (progesterone, estrogeni, androgeni), o sono coinvolti in funzioni omeostatiche modulando equilibri idrico-salini (mineralcorticoidi), o i livelli ematici di glucosio, le risposte immunitarie, le risposte allo stress (glucocorticoidi), ecc. Nell'esplicare queste funzioni, gli ormoni steroidei agiscono a livello delle cellule bersaglio tramite proteine recettoriali specifiche, che modulano l'espressione di una grande varietà di geni specifici, agendo come fattori di trascrizione. I recettori per il progesterone, gli estrogeni, gli androgeni, i glucocorticoidi e i mineralcorticoidi sono stati caratterizzati sia biochimicamente che, più recentemente, con metodi di biologia molecolare che hanno permesso il clonaggio del DNA che codifica per le varie proteine. Attraverso i dati raccolti, si è definita una famiglia di proteine con caratteristiche comuni, sia in termini strutturali (per la presenza di caratteristici domini funzionali), sia per il meccanismo d'azione, che è generalmente conosciuta come "famiglia dei recettori degli steroidi"; questa super-famiglia comprende anche proteine recettoriali il cui legante è solo parzialmente correlato, dal punti di vista strutturale, agli steroidi, come la vitamina D, o la cui struttura è totalmente non-steroidea come gli ormoni tiroidei, o l'acido retinoico (Evans, 1988). Infine comprende anche una serie di proteine, caratterizzate durante il clonaggio dei vari recettori per gli steroidi che hanno alcune caratteristiche in comune con detti recettori, ma che vengono chiamate orfane in quanto non è stato per loro ancora identificato nessun tipo di legante (Carson-Jurica et al., 1990). Il lavoro qui descritto riguarda un aspetto particolare del meccanismo d'azione del recettore steroideo e cioè la sua fosforilazione. Verrà discusso con maggiore attenzione quello che avviene nel caso del recettore per il progesterone; si cercherà tuttavia di evidenziare, ove possibile, e sulla base della letteratura disponibile, i meccanismi comuni a tutta la famiglia dei recettori steroidei.

Introduction: Cellular retinoic acid metabolism and homeostasis; Epigenetic gene regulation by retinoic acid; Retinoic acid signalling in breast development and morphogenesis; Disruption of epigenetic retinoic acid signalling in breast tumorigenesis. Scope of the thesis Materials and Methods Experimental Work: An impaired RA signal leads to RARbeta2 epigenetic silencing and RA resistance; Impairment of RA-RARalfa signalling leads to the concerted epigenetic silencing of RARbeta2 and CRBP1 in untransformed human breast epithelial cells; Mechanistic relationship between RARbeta2 and CRBP1 transcription and different phenotypes of breast epithelial cell transformation: Implications for predicting breast cancer risk; CRABP2 downregulation results in epigenetic dysregulation of RA signalling and consequent breast epithelial cell transformation. General Discussion Summary References Curriculum Vitae

Background: The p.E318K variant of the microphthalmia-associated transcription factor (MITF) has been implicated in genetic predisposition to melanoma as an intermediate penetrance allele. However, the impact of this variant on clinico-phenotypic, as well as on dermoscopic patterns features of affected patients is not entirely defined. The purpose of our study was to assess the association between the p.E318K germline variant and clinic-phenotypical features of MITF+ compared to non-carriers (MITF-), including dermoscopic findings of melanomas and dysplastic nevi. Methods: we retrospectively analyzed a consecutive series of 1386 patients recruited between 2000 and 2017 who underwent genetic testing for CDKN2A, CDK4, MC1R and MITF germline variants in our laboratory for diagnostic/research purposes. The patients were probands of melanoma-prone families and apparently sporadic single or multiple primary melanoma patients. For all, we collected clinical, pathological information and dermoscopic images of the histopathologically diagnosed melanomas and dysplastic nevi, when available. Results: After excluding patients positive for CDKN2A/CDK4 pathogenic variants and those affected by non-cutaneous melanomas, our study cohort comprised 984 cutaneous melanoma patients, 22 MITF+ and 962 MITF-. MITF+ were more likely to develop dysplastic nevi and multiple primary melanomas. Nodular melanoma was more common in MITF+ patients (32% compared to 19% in MITF-). MITF+ patients showed more frequently dysplastic nevi and melanomas with uncommon dermoscopic patterns (unspecific), as opposed to MITF- patients, whose most prevalent pattern was the multicomponent. Conclusions: MITF+ patients tend to develop melanomas and dysplastic nevi with histopathological features, frequency and dermoscopic patterns often different from those prevalent in MITF- patients. Our results emphasize the importance of melanoma prevention programs for MITF+ patients, including dermatologic surveillance with digital follow-up.

Mycosis fungoides (MF) and Sézary syndrome (SS) are two variants of cutaneous T-cell lymphomas (CTCL). These entities are considered two distinct diseases, based on phenotypic and genetic analyses. Neoplastic cells in MF seem to derive from T effector memory cells, instead, neoplastic SS cells have a central memory (TCM) profile. Moreover, evidence of a resident memory T cell (TRM) profile in early-stage MF, compared to migratory memory T cells (TMM) profile in advanced-stage MF, seems to explain the clinical behavior of MF during progression. Recent studies have shown more heterogeneity in CTCL phenotypes compared to that theory. Differences between MF and SS also involve the microenvironment, which predominantly expresses Th1 phenotype compared to Th2 phenotype in advanced stages and SS. A series of studies have identified multiple molecular changes in CTCL, showing a heterogeneous landscape of numerous genetic alterations without a strong differential signature between the two diseases. Molecular mutations could be found more frequently in the pathways of epigenetic and/or chromatin regulation, TCR and T-cell/ cytokine signaling, JAK/STAT signaling, and phosphoinositide 3-kinases (PI3K)/protein kinase B (Akt) and NF-kB pathway. The aim of this project is to delineate the differences between MF and SS about the phenotypic and genetic point of view, taking into account all stages of the diseases. In the first phase of the project, 15 patients with a new diagnosis of MF and SS were selected, including 5 early-stage MF (IA-IIA), 3 advanced MF (2 III MF, 1 IIB MF), 5 classical SS, 2 non-erythrodermic SS. Neoplastic T-cell immune phenotypes were evaluated on paraffin-embedded formalin-fixed sections of skin biopsies and on CD4+CD7- sorted T cells of the peripheral blood by flow cytometry (except for two CD7+ cases). About the 3 patients with stage IB MF, two of them revealed a TRM phenotype (CD69+CD103+CCR7-CD62L-). In the third of them, T cells aberrantly showed expression of CD69 and also CD62L. One IIA-MF patient showed expression of CD69 and CD103 (TRM markers) but also a partial expression of CCR7 with the negativity of CD62L, as TMM phenotype. Erythrodermic and tumoral MF patients were characterized by an infiltrate with a TMM phenotype. In SS, 3 patients showed a typical TCM phenotype (CD45RO+CD27+CCR7+CD62L+). Out of 5 CD45RA+ cases, 4 of them evidenced a T naïve phenotype (CD62L+), including an early MF (IIA). One SS showed complete negativity of CD62L and CCR7, arguing a TEMRA phenotype. Immunophenotype of blood samples revealed a better correlation with the skin of SS patients compared to MF patients. Neoplastic T cells in MF mainly had a Th1 phenotype, also in advanced stages, compared to Th2 phenotype of SS patients. This first step of the project confirmed that MF and SS are characterized by heterogeneity of phenotypes with partial correlation to the clinical features. Evidence of the same TCM phenotype in patients with classical and atypical SS suggests that criteria of this disease should be revised, including also non-erythrodermic forms. In the second phase of the project, we performed a gene expression analysis using a 770-genes panel by NanoString technologies (PanCancer Immune Profiling Panel). To obtain more statically significant data, we included 95 FFPE slides of CTCL. Total RNA from 87 samples was extracted (36 SS and 51 MF at any stage). NanoString data were processed and statistical analysis was performed within the statistical environment R. A principal component analysis was performed on all samples together, but we found a homogeneous group without any evidence of clustering between MF and SS. So, we decided to study separately the two entities. About MF, 12 differentially expressed genes (DEGs) between advanced stages and early stages were found (p-value <0.01): 9 of them resulted upregulated (CCR3, PRAME, FPR2, PMCH, AMBP, TRL7, TNFRSF10C, CFI, HAVCR2) and 3 were down-regulated (KLRB1, CD1B, CD5). Gene ontology (GO) enrichment analysis showed that those genes were significantly enriched in the regulation of the immune system, macrophage activation and Toll-like receptor (TLR) signaling. (FDR <0.05) KEGG pathways analysis revealed a not significantly representation in any pathways. Then, MF cohort was divided into two groups based on the median expression for each gene and the effects of high or low expression levels on OS were examined using the Kaplan-Meier (KM) survival curve. A list of 39 genes was identified as significant associated with OS (p-value <0.05). The HRs and p-values of those selected 39 genes were calculated through Cox regression model, revealing a 9-genes signature, which did not match with DEGs but showed a high significance from KM survival analysis. The increased expression of the following genes is significantly associated with poor prognosis: CDK1 (HR=2.06), IL6ST (HR=1.49), CCR4 (HR=1.66), ITK (HR=1.78), NOS2A (HR=1.38), IL2RA (HR=2.06), LRRN3 (HR=1.39), DUSP4 (HR=1.74). Instead, the lower expression of CCL26 (HR=0.53, p-value 0.028) is significantly associated to poor prognosis. A prognostic score was developed based on the incidence of each gene of the signature on overall survival and was defined as the linear combination of logarithmically transformed gene expression levels weighted by average Cox regression co-efficient. Patients with a high score of expression of the signature had a significantly poorer prognosis compared to patients with a low score (p-value < 0.05). Similarly, considering advanced versus early stages, the two groups with high score confirmed to have a statistically significant poorer prognosis compared to low score (p-value <0.05). This result is particularly evident comparing the score in early stages. KEGG pathway analysis showed that the 9-genes signature was significantly enriched in cytokine and JAK-STAT signaling. For SS samples, we did not evaluate DEGs because of the absence of different stages like in MF, but we performed the same analyses based on OS. A 14-gene signature was found, characterized by the association of their high expression and poor prognosis. The genes identified were IL12A (HR=2.12), IL5RA (HR=2.80), IFNL2 (HR=2.21), NT5E (HR=1.97), IL18RAP (HR=1.89), ABCB1 (HR=1.74), CCL16 (HR=1.89), CCL1 (HR=1.95), IL22RA2 (HR=1.67), IFNA17 (HR=1.69), C9 (HR=1.79), CCR9 (HR=3.31), SPANXB1 (HR=1.62), TREM1 (HR=1.76). We applied the prognostic score, showing that patients with a high score had a worse prognosis compared to patients with a low score. GO analysis showed that this 14-gene signature was significantly involved in defense response and Th1 cytokine production (FDR <0.05). KEGG pathway analysis was able to confirm a statistically significant involvement of these genes in JAK-STAT and TLR signaling. In this second phase of the project, we were able to find a gene signature for each disease with a significant prognostic value that could be useful in clinical practice, especially in early stages. Evidence of a strong involvement of JAK-STAT pathway in analysis of both MF and SS is interesting because this pathway is well known to be involved in CTCL pathogenesis and its pharmaceutical inhibition is still studied.

2014 - 2015
The urokinase type plasminogen activator (uPAR) is a three domain GPI-anchored cell surface receptor. uPAR expression is strongly up-regulated and represents a negative prognostic factor in various tumors, including hematologic malignancies. uPAR expression is post-transcriptionally regulated by RNA binding proteins (RBPs). RBPs bind specific sequences in the 3’untranslated region (3’UTR) of uPAR-mRNA, stabilizing or destabilizing the transcript. The 3’UTR of transcripts from a large number of genes includes target sequences also for small translational repressors RNAs (miRNAs). miRNAs play key roles in many cellular pathways; their aberrant expression is a common feature of various malignancies. We selected three miRNAs miR-146a, miR-335 and miR-622 that could bind the 3’UTR of uPAR-mRNA; these three miRNAs, as reported in literature, are expressed in CD34+ HSC or in acute myeloid leukemia (AML) cells and can act as oncosuppressors by inhibiting oncogene expression. We found that selected miRNAs regulate uPAR expression by directly targeting its 3’UTR in AML cell lines. Indeed, uPAR expression is reduced by their overexpression and increased by their specific inhibitors. Overexpression of selected miRNAs impaired cell migration, invasion and proliferation of AML cell lines. Interestingly, we found an inverse relationship between uPAR expression and miR- 146a and miR-335 levels in AML blasts. This suggests their possible role in regulating uPAR expression also in vivo. We also investigated the capability of uPAR-3’UTR to act as competing endogenous RNA (ceRNA). We showed that uPAR-3’UTR overexpression up-regulates uPAR expression and expression of other targets of selected miRNAs; these results suggest that uPAR-3’UTR may recruit selected miRNAs, allowing translation of their targets, thus acting as ceRNA. [edited by Author]
XIV n.s.

Intrauterine Growth Restriction (IUGR) is a pregnancy-related pathology characterized by a placental insufficiency phenotype and a multifactorial etiology that still needs to be completely clarified. IUGR is associated with increased risk of maternal and neonatal perinatal mortality and morbidity and a tendency to develop cardiovascular and metabolic pathologies in the adulthood. A deeper knowledge of the alterations occurring in IUGR has therefore become essential to find therapeutic tools to prevent fetal, neonatal and future adult complications. A specific placental phenotype has been associated with IUGR, characterized by placentation defects, altered transport of oxygen and nutrients to the fetus, impaired mitochondria content and increased oxidative stress (OxS). Mitochondria (mt) are eukaryotic ubiquitous organelles whose number range from hundreds to thousands of copies per cell. As they are the fuel stations of all cells, more than 95% of ATP is synthesized in these organelles Besides this well-known function, many essential pathways involve mitochondria, such as mt biogenesis. Mt biogenesis is a complex of mechanisms needed to mitochondria ex-novo creation: mt DNA duplication and translation of mt factors controlling the transcription machinery that produce all respiratory chain complexes (RCC). IUGR hypoxic features, and the consequent higher OxS, affect mitochondria as showed by in vivo models increased mt oxygen consumption trigger by hypoxia or in vitro downregulation of mt biogenesis. The aim of this study was to investigate, by ex vivo experiments and in vitro models, different types of placental cells to deeper characterize the placental insufficiency features of IUGR, with specific attention to the consequences of its hypoxic environment. IUGR and physiological placenta bioenergetics were first examined, by analyzing both mitochondrial (mt) content and function in whole placental tissue and in several placental cell types (cytotrophoblast and mesenchymal stromal cells). Mt DNA content resulted higher in IUGR placentas compared to controls, as well as NRF1 (biogenesis activator) mRNA levels. Oppositely, both mtDNA and NRF1 expression levels were significantly lower in cytotrophoblast cells isolated from IUGR placentas compared to controls. The observed divergence between placental tissue and cytotrophoblast cells may suggest that other placental cell types (e.g. syncytiotrophoblast, endothelial cells and mesenchymal stromal cells), that are subjected to different oxygen - and consequently oxidative stress - levels may be responsible for the mt content increase in the whole placental tissue. Moreover, a different exposure to progesterone may also explain this mt content divergence, since progesterone, regulating mt biogenesis, is produced by syncytio but not in cytotrophoblast cells. In IUGR cytotrophoblast cells, respiratory chain complexes (RCC) showed lower, though not significantly, gene expression levels and no differences in their protein expression compared to controls. In contrast, mt bioenergetics - represented by cellular O2 consumption - was higher in IUGR versus controls, especially in more severe IUGR cases. Thus, despite the protein content of RCC was not altered, their activity was significantly increased in IUGR cytotrophoblast cells, possibly due to a more efficient RCC assembly. Finally, as O2 consumption resulted inversely correlated to mtDNA in cytotrophoblast cells, a functional (respiration) compensatory effect to the decreased mitochondrial content might be hypothesized. Estrogen-Related Receptor (ERRγ) is a very interesting transcriptional factor involved both in mt biogenesis and function and in estradiol production (through CYP19 aromatase up-regulation). ERRγ and CYP19 mRNA levels were therefore analyzed, for the first time in human IUGR placentas. In whole placental tissue CYP19 showed higher expression in IUGR compared to controls, progressively increasing with IUGR severity. Higher ERRγ expression in IUGR cases was also found, though not significantly. These data are consistent with mtDNA and NRF1 results, thus confirming altered mt biogenesis and content in IUGR and strengthening the hypothesis of a restore attempt made through the stimulation of mt biogenesis. An additional effect of ERRγ increase is CYP19 upregulation. The observed higher CYP19 expression may indicate a protective mechanism exerted through estradiol against oxidative stress. Opposite to their placental tissue expression, ERRγ levels in cytotrophoblast cells significantly decreased in the IUGR group compared to controls. This is consistent with literature evidences of O2-dependent ERRγ gene expression in trophoblast cells. As well as for mt DNA and NRF1 levels, other cell types could be responsible for ERRγ increase in the whole placental tissue. CYP19 expression was not significantly different between IUGR and controls in cytotrophoblast cells, though it positively correlates with ERRγ levels, but low CYP19 levels are reported for cytotrophoblast cells, and this might complicate the detection of any difference. Interestingly, a significant positive correlation linked maternal BMI and expression of both ERRγ and CYP19 genes (in whole placental tissue: positive trend/cytotrophoblast cells: negative trend). An estradiol-dependent regulation of leptin production through ER (Estrogen Receptor) – ERR is known. Leptin, an anti-obesity hormone produced also by placenta, increase during. The future measure of plasmatic levels of both leptin and 17β estradiol in maternal blood will verify this speculation. Then in vitro experiments were performed to assess possible biomolecular mechanisms regulating mithocondrial content in Intrauterine Growth Restriction, by culturing primary placental cells under normal oxygen conditions and hypoxia, a typical feature of IUGR. Fluctuations in placental oxygen concentration may generate oxidative stress (OxS), that is enhanced in Intrauterine Growth Restriction condition. As mitochondria are the major producers of intracellular reactive oxygen (O2) species through free radicals generated by the mt oxidative phosphorylation, altered intrauterine O2 conditions might affect mt DNA content and function, leading to increased oxidative stress in IUGR placental cells. Using trophoblast primary cell lines could help to understand O2 conditions that placentas may be exposed to in IUGR pregnancy. Exposure of trophoblast cultures to hypoxia is an in vitro model commonly used in the last few years. Preliminary data from performed experiments show that the oxygen lack in cytotrophoblast cells leads to increased mt DNA levels. The evidence that O2 levels may regulate mt biogenesis in cytotrophoblast cells highlights their deep sensitivity to O2 conditions. However, further data are needed to confirm these preliminary results, also considering the implied difficulties in adapting the primary cytotrophoblast cultures, very sensitive to O2 concentration, to an in vitro model. A future goal will be reproduce particularly hypoxia/re-oxygenation intervals characterizing placental insufficiency and generating OxS and measuring cell apoptosis levels and autophagy markers (e.g. TNF-α, p53, caspases). Finally, in vitro experiments were performed to isolate and characterized p-MSCs from physiological and affected by IUGR placentas. p-MSCs have never been investigated before in IUGR pregnancies, but their role have been recently studied in preeclamptic placentas. PE p-MSCs show pro-inflammatory and anti-angiogenic features, that may result in abnormal placental development. In the performed p-MSCs cultures, mesenchymal markers enrichment and multipotent differentiation abilities confirm the successful isolation and selection of a mesenchymal stromal cell from placental membranes and basal disc of both physiological and IUGR placentas. As attested by flow cytometry data, the p-MSC population is earlier selected in IUGR placentas: this faster selection might represent a compensatory mechanism to metabolic alterations occurring in IUGR placental cells and/or to the adverse IUGR placental environment. During placenta development, the lower proliferation rate characterizing IUGR pMSCs could impair the primary villi formation and consequently trophoblast development, since MSCs both serve as structural of trophoblast cells. Moreover, IUGR p-MSCs population display lower endothelial and higher adipogenic differentiation potentials compared to controls. During pregnancy, pMSCs usually contribute to both vasculogenesis and angiogenesis Interestingly, several studies report some alterations in maternal and fetal endothelial progenitor or in the angiogenic capacity of IUGR placental cells. Opposite to endothelial differentiation ability, the adipogenic potential in pMSCs from IUGR is increased compared to controls: as these changes are evident early in life, the predisposition to obesity may be programmed in utero. To further characterize IUGR pMSCs, their mitochondrial (mt) content was investigated by measuring NRF1 and Respiratory Chain UQCRC1 and COX4I1 gene expression levels. Mesenchymal stem cell metabolism is known to be mainly anaerobic, with a shift towards an aerobic mitochondrial metabolism reported during differentiation. Interestingly, p-MSCs cultured with no differentiating medium present a trend towards higher NRF1, UQCRC1 and COX4I1 expression levels in IUGR basal disc samples compared to controls and higher COX4I1 levels in IUGR placental membranes; these differences are not statistically significant likely because of the low sample number. Nevertheless, they might account for metabolic alterations in IUGR p-MSCs, showing a possible shift to aerobic metabolism, with the loss of the metabolic characteristics that are typical of multipotent and undifferentiated cells. The different gestational age between cases and controls, typical of all IUGR versus term-placentas studies, is a possible limit that associate all the performed experiments. However, any significant correlation between gestational age (ge) and the O2 consumption of CIV (which presents the highest significance between IUGR and controls), ge and mt DNA levels, ge and ERRy/CYP19 expression, ge and p-MSCs. CYP19 gene expression have been analyzed assuming that it may represent an index of aromatase content in placental tissue. However, post-translational modifications (glycosylation and phosphorylation) may occur, affecting its functional activity. Finally, a potential limitation of placental mesenchymal stromal cells is that the analysis was performed on IUGR placentas at delivery, whereas placental abnormal development of IUGR pathology is supposed to start already at the beginning of placentation. Taken together, reported data highlight mitochondrial alterations occurring in placentas of Intrauterine Growth Restricted pregnancies, through ex vivo and in vitro approaches. These results shed genuine new data into the complex physiology of placental oxygenation in IUGR fetuses. Mitochondrial content is higher in IUGR total placental tissue compared with normal pregnancies at term. This difference is reversed in cytotrophoblast cells of IUGR fetuses, which instead present higher mitochondrial functionality. These findings suggest different mitochondrial features depending on the placental cell lineage. Indeed, our results on placental Mesenchymal Stromal Cells, showed higher levels of genes accounting for mitohcondrial content and function. The increased placental O2 consumption by placental tissue may represent a limiting step in fetal growth restriction, preventing adequate O2 delivery to the fetus. This limitation has potential consequences on fetal O2 consumption both in animal models and in human IUGR.

Recent advances in 3D culture technology allow embryonic and adult mammalian stem cells to generate organoids in vitro, which reflect key structural and functional properties of organs they originate (Clevers H., Cell, 2016). For this reason, they represent a powerful tool to study human physiological and pathological processes, in particular to investigate complex processes like tumorigenesis and tumor growth, resembling the in vivo mechanisms. In particular, in tumor contest neoplastic cells activate several strategies to escape from immunesurveillance, such as the recruitment of immune cells with immunosuppressive functions. In this regard, CD4+ T regulatory cells (Tregs), physiologically engaged in the maintenance of immunological self-tolerance and immune homeostasis, are potent suppressors of effector cells and found at high frequencies in various types of cancer. A recent transcriptome analysis performed in our lab (De Simone M. et al., Immunity, 2016) revealed that tumor-infiltrating Tregs, isolated from CRC (colorectal cancer) and NSCLC (non-small cell lung cancer) patients, expressed a unique and specific gene signature, correlated with patients’ survival. In line with our findings, non-lymphoid tissue infiltrating Tregs can exhibit specific phenotypes and transcriptional profile involved in glucose metabolism, tissue repair and muscle regeneration, far from their well-established suppressive roles (Cipolletta D. et al., 2012; Arpaia N. et al., 2015). Thus, our work wants to evaluate the immune dependent and independent function of tissue- infiltrating Tregs, exploiting a co-culture model with normal and colon cancer- derived organoids. This approach could be suitable to recapitulate primary tumorigenesis, cancer microenvironment effect on Tregs recruitment and phenotype and, vice versa, infiltrating Tregs influence on tumor onset, growth and tissue homeostasis. In order to answer our biological questions by using organoid model, we first of all derived organoids starting from CRC patients’ biopsies, according to protocol published by Sato T. et al. (2011). We generated a biobank of 20 and 28 human- normal and tumoral colon- derived organoid lines, respectively, from tumoral biopsies and the adjacent normal mucosa of patients affected by colorectal cancer. In particular, these organoid lines can be propagated, frozen and defrosted like any tumour cell line, morphologically recapitulating the cellular composition and architecture of colon primary tissue and, importantly, representative of all CRC molecular subtypes. Moreover, our RNA-seq bulk analysis on our organoid lines unveiled that they are very stable from the transcriptional point of view during passages and preserve the inter-individual heterogeneity. Furthermore, our ChIP-seq data showed that our library resembled the same epigenetic landscape of colorectal primary tumour; in this context our study represents an innovative approach to investigate epigenetic processes leading CRC development and progression. Considering that Tumor-infiltrating- Tregs (TI- Tregs) were found to express a peculiar gene signature (De Simone et al., 2016; Plitas et al., 2016), we decided to exploit organoid model to better elucidate processes leading the development and the recruitment of these cells at the tumour site. To this end, we proceeded with the establishment of Tregs- PDO co-culture, assessing the feasibility of this system and evaluating Tregs viability in organoid culture conditions, without observing any significant differences in their survival. Moreover, we evaluated their ability to migrate into 3D organoid structure, revealing their capacity to overcome firstly the obstacle represented by Matrigel (which mimics extracellular matrix) and then creeping into the 3D architecture of organoids, recapitulating the same behaviour they have when recruited at the tumour site. An important evidence about the possible influence of tumoroids on Treg phenotype came from our preliminary co-culture experiment, revealing that PDO-Tregs co-culture upregulated the expression of PDL1 (one of the genes belonging to TI-Treg signature (De Simone et al., 2016)) specifically on Treg but not on Tconv cells. On the other hand, with our co-culture preliminary experiment we observed that expanded TI-Tregs enhanced PDL1 expression on tumoroid cells, which not happened when organoids were co-cultivated with Tconv cells, suggesting the possible influence of Tregs on the expression of specific molecules on cancer cell surface. In conclusion, the exploitation of TI-Treg-PDO co-culture could shed light on the interplay between tumor and TI-Tregs, giving the opportunity to potentially interfere in this crosstalk and design a peculiar anticancer therapy targeting TI-Tregs in a personalized manner.

Background. Metabolic reprogramming is a key feature of neoplastic transformation and mitochondria are the most important organelles in such oncogenic process. Recent evidence suggests that TRAP1 is a key player in tumor-related metabolic rewiring. Most studies have addressed TRAP1- related oncogenesis by in vitro and in vivo analyses. Little is however known on the possible contribution of histology to such studies. Study aims. This study assessed the role of histology in the study of TRAP1-related metabolic reprogramming. Specifically, it aimed: (i) to integrate the results of in vitro and in vivo studies with the histological analysis of tumor samples; (ii) to verify the correspondence between primary human neoplasms and animal tumor models; (iv) to identify novel fields for the study of TRAP1-related oncogenic cascades. Materials and methods. This project considered the following neoplastic settings: (i) neurofibromatosis type 1 (NF1)-related benign and malignant nerve sheath tumors; and (ii) germinal center (GC)-derived lymphoproliferative disorders. For the NF1-related tumors, morphological analysis and phenotypic characterization (TRAP1, HIF1a and related metabolic markers) were performed on: (i) human samples of plexiform neurofibroma (PN) and malignant nerve sheath tumors (MPNST); (ii) engineered mouse models of NF1-related neoplasms; and (iii) xenografts of MPNST. For GC-derived lymphomas, TRAP1 expression was assessed in non-neoplastic lymphoid tissues and in Burkitt lymphoma (BL), diffuse large B-cell lymphoma (DLBCL) and Hodgkin lymphoma (HL) samples. The immunohistochemical results were integrated with the results of in silico gene expression studies (Oncomine database). Results. Histological analysis of human PNs and MPNSTs documented the expression of TRAP1, HIF1a and downstream metabolic markers in both benign and malignant samples. A progressive increase in the positivity for such proteins was noted along the oncogenic cascade from nonneoplastic nerves to benign (PN) and malignant (MPNST) tumors. Similar expression patterns were observed in the animal tumor models. In this context, histological evaluation also proved instrumental: (i) to confirm the correspondence between human and animal tumors; (ii) to investigate the metastatic potential of MPNST xenografts; (iii) to detect the effects of TRAP1 knock-down on tumor cell growth and metabolic reprograming; and (iv) to highlight strongly versus minimally activated metabolic pathways in NF1-related oncogenic cascades. The histological characterization of reactive lymphoid tissues highlighted TRAP1 expression in subsets of GC blasts (i.e. differentiating immunoblasts and re-cycling centroblasts). The joint expression of TRAP1 and HIF1a in GC blasts confirmed the presence and activation of the TRAP1/HIF1a axis in GC physiology. In silico studies of GC-derived lymphomas showed very high TRAP1 mRNA levels in BL and (to a lesser extent) DLBCL and HL. Immunohistochemical analysis of primary tumor samples confirmed the in silico results. Conclusions. Histological analysis contributes to the understanding of tumor metabolism and integrates the results of in vitro and in vivo biochemical studies. In particular, it confirms the relevance of TRAP1 activation in NF1-related peripheral nerve sheath tumors and discloses a tight correspondence between primary human samples and animal tumor models. Immunohistochemical characterization of reactive lymphoid tissues and primary lymphoma samples also identifies specific TRAP1 expression profiles, possibly subtending tumor-related metabolic networks.
Background. Metabolic reprogramming is a key feature of neoplastic transformation and mitochondria are the most important organelles in such oncogenic process. Recent evidence suggests that TRAP1 is a key player in tumor-related metabolic rewiring. Most studies have addressed TRAP1- related oncogenesis by in vitro and in vivo analyses. Little is however known on the possible contribution of histology to such studies. Study aims. This study assessed the role of histology in the study of TRAP1-related metabolic reprogramming. Specifically, it aimed: (i) to integrate the results of in vitro and in vivo studies with the histological analysis of tumor samples; (ii) to verify the correspondence between primary human neoplasms and animal tumor models; (iv) to identify novel fields for the study of TRAP1-related oncogenic cascades. Materials and methods. This project considered the following neoplastic settings: (i) neurofibromatosis type 1 (NF1)-related benign and malignant nerve sheath tumors; and (ii) germinal center (GC)-derived lymphoproliferative disorders. For the NF1-related tumors, morphological analysis and phenotypic characterization (TRAP1, HIF1a and related metabolic markers) were performed on: (i) human samples of plexiform neurofibroma (PN) and malignant nerve sheath tumors (MPNST); (ii) engineered mouse models of NF1-related neoplasms; and (iii) xenografts of MPNST. For GC-derived lymphomas, TRAP1 expression was assessed in non-neoplastic lymphoid tissues and in Burkitt lymphoma (BL), diffuse large B-cell lymphoma (DLBCL) and Hodgkin lymphoma (HL) samples. The immunohistochemical results were integrated with the results of in silico gene expression studies (Oncomine database). Results. Histological analysis of human PNs and MPNSTs documented the expression of TRAP1, HIF1a and downstream metabolic markers in both benign and malignant samples. A progressive increase in the positivity for such proteins was noted along the oncogenic cascade from nonneoplastic nerves to benign (PN) and malignant (MPNST) tumors. Similar expression patterns were observed in the animal tumor models. In this context, histological evaluation also proved instrumental: (i) to confirm the correspondence between human and animal tumors; (ii) to investigate the metastatic potential of MPNST xenografts; (iii) to detect the effects of TRAP1 knock-down on tumor cell growth and metabolic reprograming; and (iv) to highlight strongly versus minimally activated metabolic pathways in NF1-related oncogenic cascades. The histological characterization of reactive lymphoid tissues highlighted TRAP1 expression in subsets of GC blasts (i.e. differentiating immunoblasts and re-cycling centroblasts). The joint expression of TRAP1 and HIF1a in GC blasts confirmed the presence and activation of the TRAP1/HIF1a axis in GC physiology. In silico studies of GC-derived lymphomas showed very high TRAP1 mRNA levels in BL and (to a lesser extent) DLBCL and HL. Immunohistochemical analysis of primary tumor samples confirmed the in silico results. Conclusions. Histological analysis contributes to the understanding of tumor metabolism and integrates the results of in vitro and in vivo biochemical studies. In particular, it confirms the relevance of TRAP1 activation in NF1-related peripheral nerve sheath tumors and discloses a tight correspondence between primary human samples and animal tumor models. Immunohistochemical characterization of reactive lymphoid tissues and primary lymphoma samples also identifies specific TRAP1 expression profiles, possibly subtending tumor-related metabolic networks.

La lamina nucleare (NL) è un reticolo di proteine fibrillari che riveste la superficie interna della membrana nucleare. Essa è principalmente composta da filamenti intermedi di tipo V , chiamati lamìne, e proteine ausiliari ad esse associate (lamin-associated proteins, LAPs). Nei mammiferi le lamìne sono codificate da tre geni : LMNA, che codifica per la lamìna A e lamìna C (lamìne di tipo A), LMNB1 e LMNB2 che codificano rispettivamente per la lamìna B1 e B2 (lamìne di tipo B). Lamìne e LAPs si associano a formare una matrice tridimensionale densa e dinamica che stabilisce numerose interazioni, sia stabili che transitorie, con diverse classi di molecole biologiche: DNA, fattori di trascrizione, proteine strutturali. Tutte queste interazioni sono essenziali per fornire stabilità strutturale e preservare l’integrità nucleare, per collegare fisicamente e funzionalmente la lamina nucleare al citoscheletro e per organizzare la cromatina. In questo modo, oltre a svolgere un ruolo strutturale fondamentale, la lamina nucleare risulta ricoprire ruoli chiave anche nei processi di meccanotrasduzione del segnale e nella regolazione dell’espressione genica ed epigenetica. Mutazioni a carico dei geni che codificano per le lamìne nucleari sono associate ad un'ampia ed etogenea classe di patologie note come laminopatie. Tra queste, una delle più controverse ed interessanti è la Hutchison-Gillford Progeria Syndrome (HGPS), una malattia genetica rara dovuta a una mutazione puntiforme nel gene LMNA. Tale mutazione risulta nella produzione di una versione tronca della lamìna A, mancante di 50 amminoacidi, conosciuta come Progerina. HGPS è principalmente caratterizzata da alterazioni morfologiche del nucleo e invecchiamento precoce. I soggetti affetti da Progeria, infatti, fin dai primi anni di vita sviluppano condizioni patologiche tipiche dell’età senile quali cataratta, diabete e l'osteoporosi pur preservando le normali funzioni congnitive. Questi pazienti muoiono tipicamente per complicanze cardiovascolari intorno ai 14 anni di età, in media. Considerando la sindrome di Hutchinson-Guilford come un esempio estremo di ciò che alterazioni della lamina nucleare comportano, durante il mio dottorato di ricerca ho investigato diversi aspetti riguardanti la biologia della lamina nucleare con particolare interesse all'impatto che perturbazioni strutturali della lamina nucleare possono avere sulle normali funzioni cellulari, la meccanica cellulare, la regolazione dell'espressione genica e l’ interconnessione esistente tra integrità della lamina nucleare, processo di invecchiamento e stress ossidativo. Per ottenere una visione d’insieme del contributo della lamina nucleare sia in condizioni fisiologiche che patologiche, sono state adottate strategie di ricerca basate su approcci interdisciplinari e integrativi in grado di tenere conto degli aspetti strutturali, meccanici e molecolari. Per fa questo, in prima instanza sono state effetuate delle analisi bionformatiche: tutti i dati di trascrittomica relativi a pazienti HGPS, presenti in database pubblici e in letteratura, sono stati raccolti e analizzati rispetto a dati equivalenti ottenuti da controlli sani. Tale analisi ha permesso di delineare profilo di espressione genica tipico di pazienti HGPS e di individuare i pathways deregolati in presenza della patologia. È stato inoltre studiato l’impatto che alterazioni della lamina nucleare hanno sulle connessioni fisiche e funzionali che questa stabilisce sia con elementi nucleari ed extra-nucleari, in un modello cellulare in cui è possibile indurre sperimentalmente, in modo controllato, l’espressione della forma mutata di Lamìn A responsabile dell’ HGPS. Tale modello cellulare ricapitola fedelmente il peculiare fenotipo cellulare dei pazienti risultando essere una valida alternativa all’utilizzo di linee primarie derivanti dai pazienti. Infine, l'interdipendenza tra stress ossidativo, invecchiamento e lamìne nucleari è stata investigata in un nuovo modello cellulare di stress ossidativo sviluppato nel nostro laboratorio, efficiente nel ricapitolare il processo di invecchiamento, in vitro.
ABSTRACT The nuclear lamina (NL) is a fibrillary protein network lining the inner surface of the nuclear envelope. It is mainly composed by type V intermediate filaments called lamins and lamin-associated proteins. Three lamin genes are present in Mammals: LMNA, which encodes lamin A and lamin C (Atype lamins), as well as, LMNB1 and LMNB2 that encode lamin B1 and B2 (B-type lamins) respectively. Lamins and LAPs associate to form a dense and dynamic three-dimensional matrix that establishes a huge number of stable and transient interactions with different classes of molecules: DNA, transcription factors, nuclear pore complexes and structural proteins of the cytoskeleton. All of these interactions are essential to provide nuclear structural stability and integrity, to physically and functionally link nuclear lamina to the cytoskeleton and to organizes chromatin. Thus NL, in addition to play a fundamental structural role, it is also a key player in cellular mechanotransduction processes and gene expression and epigenetic regulation. Mutations in genes encoding for lamins are associated with a wide a range of diseases, named laminopathies. Among these, the most interesting one is Hutchison-Gilford Progeria Syndrome (HGPS), a rare fatal genetic disorder due to do a point mutation in LMNA. This mutation results in the production of a truncated version of lamina A, lacking 50 amino acids, known as Progerin. HGPS is mainly characterized by morphological changes in the nucleus and premature aging. HGPS patients indeed, from their first years of life, develop pathological conditions typical of the elderly such as cataracts, diabetes and osteoporosis while preserving the normal cognitive functions. These patients typically die from cardiovascular complications around 14 years of age, on average. Considering Hutchinson-Guilford Progeria Syndrome as an extreme example of what nuclear lamina aberration entails, during my PhD I investigated many aspects of nuclear lamina biology with particular regard to the impact of nuclear lamina structural perturbations on cell functions, mechanics, gene expression regulation and the interconnection existing between nuclear lamina integrity, ageing process and oxidative stress. Indeed, to gain a comprehensive picture of nuclear lamina biology in health and disease, it has been adopted interdisciplinary and integrative research strategies able to take into account structural, mechanical and molecular aspects. Bioinformatics study has been performed: public available transcriptomic data of HGPS patients have been analysed with respect of those of healthy matched controls. This analysis allowed to delineate the typical global gene expression profile of HGPS patients and to identify all the deregulated pathways in the presence of the pathology. Moreover, impacts of lamina alterations on its physical and functional connections with extra-nuclear and nuclear elements have been studied in an inducible expression cellular model of the mutated form of Lamin A responsible for HGPS. This cellular model faithfully recapitulates the peculiar cellular phenotype of the HGPS patients resulting to be a valid alternative to primary cell lines deriving from the patients.Finally, the interdependence between oxidative stress, ageing and lamins has been investigated in a novel oxidative stress cellular model developed in our laboratory, that is also efficient in recapitulating typical ageing profile.

The Cancer Stem Cell model supported the notion that leukemia was initiated and maintained in vivo by a small fraction of leukemia-initiating cells (LICs). Previous studies have suggested the involvement of Wnt signaling pathway in Acute Myeloid Leukemia (AML) by the ability to sustain the development of LICs. A novel hematopoietic stem and progenitor cell marker, monoclonal antibody AC133, recognizes the CD34bright CD38- subset of human acute myeloid leukemia cells, suggesting that it may be an early marker for the LICs. During the first part of my phD program we previously evaluated the ability of leukemic AC133+ fraction, to perform engraftment following to xenotransplantation in immunodeficient mouse model Rag2-/-γc-/-. The results showed that the surface marker AC133 is able to enrich for the cell fraction that contains the LICs. In consideration of our previously reported data, derived from the expression profiling analysis performed in normal (n=10) and leukemic (n=33) human long-term reconstituting AC133+ cells, we revealed that the ligand-dependent Wnt signaling is induced in AML through a diffuse expression and release of WNT10B, a hematopoietic stem cells regenerative-associated molecule. In situ detection performed on bone marrow biopsies of AML patients, showed the activation of the Wnt pathway, through the concomitant presence of the ligand WNT10B and of the active dephosphorylated β-catenin form, suggesting an autocrine / paracrine-type ligand-dependent activation mechanism. In consideration of the link between hematopoietic regeneration and developmental signaling, we transplanted primary AC133+ AML A46 cells into developing zebrafish. This biosensor model revealed the formation of ectopic structures by activation of dorsal organizer markers that act downstream of the Wnt pathway. These results suggested that the misappropriating Wnt associated functions can promote pathological stem cell-like regeneration responsiveness. The analyses performed in situ retained information on the cellular localization, enabling determination of the activity status of individual cells and allowing the tumor environment view. Taking this issue into consideration, during the second part of my phD program, I set up the application of a new in situ method for localized detection and genotyping of individual transcripts directly in cells and tissues. The mRNA in situ detection technique is based on padlock probes ligation and target priming rolling circle amplification allowing the single nucleotide resolution in heterogenous tissues. The mRNA in situ detection performed on bone marrow biopsies derived from AML patients, showed a diffuse localization pattern of WNT10B molecule in the tissue. Conversely, only the AC133bright cell population shows the Wnt signaling activation signature represented by the cytoplasmatic accumulation and nuclear translocation of the active form of β-catenin. In spite of this, we previously evidenced that the regenerative function of WNT signaling pathway is defined by the up-regulation of WNT10B, WNT10A, WNT2B and WNT6 loci, we identified the WNT10B as a major locus associated with the regenerative function and over-expressed by all AML patients. By the molecular evaluation of the WNT10B transcript, we isolated an aberrant splicing variant (WNT10BIVS1), that identify Non Core-Binding Factor Leukemia (NCBFL) class and whose potential role is discussed. Moreover, we demonstrate that the function of "leukemia stem cell", present in the cell population enriched for the marker AC133bright, is strictly related to regenerative function associated with WNT signaling, defining the key role of WNT10B ligand as a specific molecular marker for leuchemogenesis. This thesis defines the new suitable approaches to characterize the leukemia-initiating cells (LICs) and suggest the role of WNT10B as a new suitable target for AML.

ABSTRACT Background: Spinal muscular atrophy (SMA) is a neurodegenerative disease and the leading genetic cause of death during childhood. SMA is caused in the majority of the cases (up to 95%) by mutations in the Survival Motor Neuron 1 (SMN1) gene coding for the SMN protein, resulting in a progressive muscular paralysis due to lower motor neurons degeneration. A deeper knowledge of SMN biology and of its role in different organs/system in reliable models is very important for the optimization of available treatments and the development of complementary therapeutic approaches. In particular, it can be crucial to generate a human model able to recapitulate the complexity of the central nervous system (CNS) and its development. A promising tool to study SMA pathology is the three-dimensional (3D) organoid, obtained starting from induced pluripotent stem cell (iPSCs). Moreover, this model could be used to identify new therapeutic strategy. Rationale: In this project, we exploited CNS organoid technology, which is a novel stem cell-based 3D platform that has the potential to address the limitations of human existing bi-dimensional (2D) cultures improving preclinical testing. We aim to demonstrate that this approach can recapitulate some of the complexity of whole-organism biology overcoming the conventional use of 2D iPSCs-derived motor neurons. Nowadays, several therapeutic strategies have been tested in clinical trials and two compounds have been approved by FDA. Nevertheless, all these approaches are completely efficacious only if administered at pre-symptomatic stages. The generation of a new model for SMA could lead to a better knowledge of the mechanisms underlying the disorder during the development of the CNS and it might contribute to develop new or combined therapeutic options for affected patients. Methods: We obtained iPSCs from human fibroblasts of both healthy subjects and SMA type 1 patients and, using two different protocols that recapitulate the embryonal developmental steps, we generated 3D brain organoids and 3D spinal cord-like spheroids. We performed immunohistochemical and molecular analysis to confirm their differentiation state. Moreover, to verify their basal activity and their capability to response to stimuli, we performed calcium imaging and electrophysiological analysis. Results: CNS organoids derived from healthy subjects and patient have been successfully obtained, as suggested by the protein and gene expression data. In particular, brain organoids gave rise to an early cerebral cortex-like formation containing progenitor cells and more mature neural subtypes. Electrophysiological analysis demonstrated not only their basal activity, but also their ability to respond to stimuli. Concerning spinal cord-like spheroids, we used a modified protocol in order to induce neural caudalization and ventralization. This model gave us a powerful tool to investigate early motor neuron pathology and causes of degeneration. Like brain organoids, spinal cord-like spheroids have been characterized by immunohistochemistry, gene expression analysis and electrophysiological activity. Preliminary results suggested that SMA organoids and spheroids, compared to the controls, exhibited not only an alteration in the proper markers expression, but also in the electrophysiological activity. Conclusion: We successfully generated and characterized healthy and SMA CNS 3D organoids and spheroids that recapitulated human CNS development, showed disease-related features. This model can be used as an innovative in vitro system to study pathogenic mechanisms, identifying therapeutic targets and test potential therapeutic strategies.

The heart is the first organ to form and function in the embryo, and all subsequent events in the life of the organism depend on its function. Cardiac lineage specification and subsequent morphogenesis of the early developing heart are complex processes that rely on networks of interacting DNA-binding transcription factors and targeted activation of cardiac-specific genes. Mutations in cardiac transcription factors, the genes they regulate and the genes that regulate them, result in many inherited congenital heart defects and point to the importance of understanding the molecular basis behind these processes. Chemical alterations on DNA and histones, known as epigenetic modifications, are being increasingly studied for their importance in organogenesis, such as that of the heart. Recently, a dynamic landscape of histone modifications has been reported to occur during cardiac differentiation in vitro: distinct chromatin patterns were associated with stage-specific expression of genes functionally relevant to the heart. However, despite the growing number of reports, the role of the enzymes that catalyze these modifications remains poorly understood in cardiac differentiation in vivo. Here, we show that a definite temporal expression pattern of DOT1-like histone H3 methyltransferase (DOT1L) drives a transitional pattern of H3K79 di-methylation in the genome of differentiating cells, and that the function of this enzyme is obligatory for the correct differentiation of cardiomyocytes. In fact, we found that expression of DOT1L was increased in ex vivo embryonic and neonatal cardiomyocytes with respect to undifferentiated embryonic stem cells and adult cardiomyocytes; moreover, H3K79me2 was highly correlated with transcriptional activation in differentiating cardiomyocytes. We also found that the loci of genes expressed only in later stages of development were enriched in this activating mark, suggesting a role for H3K79me2 in the pre-activation of genes. Our results demonstrate how histone methylation, and in particular H3K79me2, regulates the transcription in developing cardiomyocytes and the central role played by DOT1L in this process. Apart from the increase in our understanding of how epigenetics controls development, our genome-wide data on H3K79me2 could lead to the identification of novel genes and transcriptional regulatory networks involved in cardiac differentiation. Altogether our study illustrates the importance of epigenetic regulation early in development to delineate the fate of a cell and in particular the role of methylation of H3K79 in cardiomyocytes stabilizing the signature for cardiac gene expression. Furthermore, this study add an important information to the intricate process of transcription activation that make a cardiomyocyte; for the first time we built an high resolution map of H3K79me2 in cardiomyocytes that could be the starting point to predict novel transcriptional regulatory networks during cardiomyocyte differentiation, as well provide the opportunity to identify novel genes that might be informative to understand developmental regulatory programs. Indeed we shed light on an additional fundamental enzyme involved in defining the epigenetic code associated with the complex process of heart development and establish a platform useful to identify new mechanisms underlying many congenital heart defects and cardiac developmental malformations.

Introduction and purposes. Multiple myeloma (MM) is a heterogeneous disease. The discovery of a class of small non-coding RNAs (miRNAs) has revealed a new level of biological complexity underlying the regulation of gene expression. It may be possible to use this interesting new biology to improve our ability to risk stratify patients in the clinic. Methods and experimental design. We performed global miRNA expression profiling analysis of 163 primary tumors included in the UK Myeloma IX clinical trial. miRNA expression profiling was carried out using Affymetrix GeneChip miRNA 2.0; expression values for 847 hsa-miRNAs were extracted using Affymetrix miRNA QC tool and RMA-normalized. There are also 153 matching samples with gene expression profiles (GEP) and 72 matching cases with genotyping data available for integrative analyses. GEP was generated on Affymetrix HG-U133 Plus 2.0 and the expression values were RMA normalized; genotyping was performed on Affymetrix GeneChip Mapping 500K Array and the copy number values were obtained using GTYPE and dChip and were inferred against normal germ-line counterpart for each sample. Results. Firstly we have defined 8 miRNAs linked to 3 Translocation Cyclin D (TC) subtypes of MM with distinct prognoses, including miR-99b/let-7e/miR-125a upregulation and miR-150/miR-155/miR-34a upregulation in unfavourable 4p16 and MAF cases respectively as well as miR-1275 upregulation and miR-138 downregulation in favourable 11q13 cases. The expression levels of the miRNA cluster miR-99b/let-7e/miR-125a at 13q13 have been shown to be associated with shorter progression free survival in our dataset. Interestingly unsupervised hierarchical clustering analysis using these 8 miRNAs identified two subclusters among 11q13 cases, which have differential effect on overall survival (OS). We then evaluated the association of miRNA expression with OS and identified 3 significantly associated miRNAs (miR-17, miR-18 and miR-886-5p) after multiple testing corrections, either per se or in concerted fashion. We went on to develop an “outcome classifier” based on the expression of two miRNAs (miR-17 and miR-886-5p), which is able to stratify patients into three risk groups (median OS 19.4 months vs 40.6 months vs 65.3 months, log-rank test P = 0.001). The robustness of the miRNA-based classifier has been validated using 1000 bootstrap replications with an estimated error rate of 1.6%. The miRNA-stratified risk groups are independent from main adverse fluorescence in situ hybridization (FISH) abnormalities (1q gain, 17p deletion and t(4;14)), International Staging System (ISS) and Myeloma IX treatment arm (intensive or non-intensive). Using the miRNA-based classifier in the context of ISS/FISH risk stratification showed that it can significantly improve the predictive power (likelihood-ratio test P = 0.0005) and this classifier is also independent from GEP-derived prognostic signatures including UAMS, IFM and Myeloma IX 6-gene signature (P < 0.002). Integrative analyses didn't show enough evidence that the miRNAs comprising the classifier were deregulated via copy number changes; however, our data supported that the mir-17~92 cluster was activated by Myc and E2F3, highlighting the potential importance of Myc/E2F/miR-17~92 negative feedback loop in myeloma pathogenesis. We developed an approach to identify the putative targets of the OS-associated miRNAs and show that they regulate a large number of genes involved in MM biology such as proliferation, apoptosis, angiogenesis and drug resistance. Conclusion. In this study we developed a simple miRNA-based classifier to stratify patients into three risk groups, which is independent from current prognostic approaches in MM such as ISS, FISH abnormalities and GEP-derived signatures. The miRNAs comprising the classifier are biologically relevant and have been shown to regulate a large number of genes involved in MM biology. This is the first report to show that miRNAs can be built into molecular diagnostic strategies for risk stratification in MM.

Although Alzheimer’s disease (AD) might be best designated as a purely degenerative disease in whose pathogenesis amyloid-beta plays a key role, it is acknowledged that in elder patients (>65years) there is an increased likelihood of other neuropathological abnormalities including cerebrovascular lesions. Over the last years, there has been accumulating evidence that the previously held sharp distinction between AD and vascular dementia (VaD) may not be so clear-cut, especially in old age. VaD is the second most common cause of dementia after AD. VaD arises as a consequence of ischemic insults such as hemorrhage and hypoperfusion that trigger neurodegeneration by depriving nerve cells of oxygen and glucose. Such deprivation results in the depletion of nerve cell energy supplies, leading to membrane depolarization, followed by an excessive release of glutamate which activates the N-methyl-D-aspartate receptor (NMDAR). This allows the influx of toxic levels of Ca2+ into nerve cells, which, in turn, activates intracellular calcium-dependent enzymes. The purine ribonucleoside adenosine (Ado) is a naturally occurring metabolite that is ubiquitously distributed throughout the body as a metabolic intermediary. Intra- and extracellular Ado levels rise in response to physiological stimuli and with metabolic/energetic perturbations, inflammatory challenges and tissue injury. The physiological responses to Ado take place as a result of the binding and activation of different transmembrane receptors: the high-affinity A1 and A2A (A2AR) receptors, the low-affinity A2B receptor, or the low-abundance A3 receptor. It has been demonstrated that A2AR is able to prevent amyloid--induced synaptotoxicity in animal models and cell cultures. Moreover A2AR has been shown to control NMDA currents and glutamate outflow in the hippocampus. Contrasting data have been reported so far on the beneficial/detrimental effects of A2AR on brain cells. The blockade of A2AR alleviates the long-term burden of brain disorders such as ischemia, epilepsy, Parkinson’s or Alzheimer’s disease. On the other hand, agonists of A2AR can protect the Central Nervous System against several insults, including ischemia and exicitotoxins. In the periphery A2AR contributes to coronary endothelial dilatation in mice, can inhibit endothelial apoptosis and preserves vascular reactivity following hemorrhagic shock in rats. Finally, increasing evidence supports the notion that A2AR is implicated in the downregulation of inflammation. In this study we evaluated the gene and protein expression of A2AR in the peripheral blood mononuclear cells (PBMCs) of patients with VaD, AD, Mild Cognitive Impairment (MCI) and healthy controls in order to investigate its potential role as an easily accessible biomarker in the differential diagnosis of dementia. This study show that A2AR expression is upregulated in the peripheral cells of a-MCI but not AD subjects, supporting an involvement of the Ado system in the early stages of AD. It also shows that A2AR expression is lower in the PBMCs of subjects with VaD than AD, highlighting its possible relevance as a biomarker that may help differentiate two forms of dementia that are often closely associated. From our results it can be concluded that A2AR may play an important but differential role in both types of dementia: its upregulation in the preclinical stages of AD could counterbalance the existing inflammatory state and its downregulation in VaD could reflect the effects of A2AR on the brain vasculature. It can therefore be suggested that A2AR could serve as a biomarker in the differential diagnosis between VaD and AD.

In recent years, the role of CD4+ regulatory T cells (Treg cells) in inhibiting the anti cancer activity of effector T cells has become increasingly evident and they are therefore currently considered promising targets for cancer immunotherapy. Despite Treg cell depletion has been reported to increase anti-tumor specific immune responses and to reduce tumor burden, some relevant issues still remain to be addressed, for a safer, more effective clinical application of these therapies. Previous findings in our lab identified unique transcriptional profiles of human Treg cells infiltrating colorectal cancer (CRC) and non-small cell lung cancer (NSCLC) supporting the existence of an underlying regulatory hubs that specifically shape tumor Treg identity and represent potential target for their functional modulation specificially in the tumor microenvironment. Treg cell transcriptome represents just a layer of the machinery that drive the acquisition of their tumor cell state. Ultimately, a comprehensive understanding of the regulatory networks that govern tumor Treg gene expression programs can provide insights on how a more selective inhibition can be achieved by interfering with the hubs that translate the cues coming from the tumor microenvironment. To define the epigenetic blueprints specific for tumor infiltrating Treg cells we have integrated distinct histone marks (H3K4me3, H3K4me1, H3K36me3, H3K27ac, H3K27me3) with ATAC-seq derived chromatin accessibility data we generated in Treg cells isolated from peripheral blood, normal and tumor tissues. To infer genome chromatin states we have employed ChromHMM a machine learning based approach that predicts the molecular structure of promoters and enhancers based on data from chromatin accessibility assays and a set of histone modification. We focused in particular on active enhancer regions, characterized by the co-presence of H3K27Ac and H3K4me1. Transcription factor footprinting analysis on the identified regulatory regions showed TF groups with distinct binding activity profile across Treg populations that are clearly lost or gained specifically in tumor Treg cells. Based on our findings we are implementing CRISPR based epigenetic modulation of selected enhancers that represent a novel strategy to reverse Treg mediated immunosuppression in the tumor microenvironment. The new knowledge coming from this project cues will lead to a better understanding of tiTreg plasticity and to the identification of novel potential targets that can guide the rational development of tiTreg reprograming and innovative therapeutics for cancer with increased efficacy and reduced adverse effects.

Hepatocellular carcinoma (HCC) is the fifth most common solid tumor and the third leading cause of cancer-related death. Current available chemotherapeutic options are not curative due in part to their resistance to conventional therapies. We have generated orthotopic HCC mouse models in immunodeficient NOD/SCID/IL2rγ null mice by injection of AFP- and/or luciferase-expressing HCC cell lines and primary cells from patients, where tumor growth and spread can be accurately monitored in a non-invasive way. Low dose metronomic administration of cyclophosphamide (LDM-CTX) causes in this model complete regression of the tumor mass with a significant increase in survival (p<0.0001), and reduction in aberrant angiogenesis, IL-6 and TNFα (but not VEGF) levels, hyperproliferation, and alteration of normal liver parenchyma, compared to untreated animals. However, the presence of residual circulating hAFP levels suggested that some tumor cells were still present in livers of treated mice. Immunohistochemistry revealed that those cells had a hAFP+/CD13+/PCNA- phenotype, suggesting that they were dormant cancer stem cells (CSC). Indeed, off-therapy mice developed rapidly tumor growth, which was still sensitive to LDM-CTX therapy. The capacity of developing hepatoshperes in vitro was drastically reduced upon LDM-CTX treatment, which resulted in selection of CD13+ cells, showing that these cells are particularly resistant to therapy. Co-treatment of the CD13-targeting drug bestatin with LDM-CTX resulted in a dramatic reduction in tumor burden. Therefore, our results demonstrate the therapeutic efficacy of administering LDM-CTX and targeting the residual CD13+ CSC-like population in these novel orthotopic HCC models, and strongly suggests that this therapy could be implemented in clinical trials.

In this thesis we studied Neuritin (also called Nrn or cpg15), a GPI-anchored protein identified among a pool of candidate plasticity-related genes induced by kainate in rat hippocampal dentate gyrus (Nedivi, Hevroni et al. 1993). It has been shown that it is involved in neurites elongation and in promoting synaptic maturation (Putz, Harwell et al. 2005). Neuritin acts in a non-cell autonomous manner to coordinately regulate the growth of apposing dendritic and axonal arbors. Its expression is regulated by neurotrophines, NGF, calcium levels and androgens. Our previous studies demonstrated that NGF up-regulates Nrn expression during PC12 neuron differentiation, and that Nrn enhances the differentiating effects of NGF on PC12 cells favoring the extension of longer neurites (Cappelletti, Galbiati et al. 2007). Interestingly, Nrn expression peaks during neuronal development and, particularly, in the embryonic proliferative zones. During embryogenesis one of the most important aspects is the active neuronal migration in order to reach the final position and obtain the correct development of the nervous system. The migration of neurons and precursor cells involves molecular mechanisms which are often similar to those involved in neurite outgrowth. The generation of the GnRH neuron network, that plays a key role in the regulation of the reproduction system, represent a particular example of neuronal migration. These neurons generates from the olfactory epithelium and move to the hypothalamus where they exerts their regulatory function. For this reason a neuronal immortalized cell line of GnRH neurons, named Gn11 cells, is considered a good model to study neuronal migration (Radovick, Wray et al. 1991; Maggi, Pimpinelli et al. 2000; Cariboni, Maggi et al. 2007). A preliminary microarray analysis performed on Gn11 cells showed neuritin as one of the most abundant genes expressed in this cell line, leading us to hypothesize that it is involved in the neuronal migration process. To demonstrate this hypothesis, we analyzed neuritin mRNA and protein expression levels in different cell lines, characterized by a different migratory behavior. Interestingly, neuritin expression is much higher in migrating than in non-migrating cells. In Boyden microchemotaxis and wound-healing assays, the migratory ability of Gn11 cells is reduced when neuritin expression is silenced, while it is increased by neuritin over-expression. We also demonstrated through a Boyden microchemotaxis assay that the over-expression of Nrn in primary culture of ganglionic eminences (the area generating the migrating interneurons) increases interneuron migration. Furthermore, our hypothesis is strenghtened by results obtained with the selective elettroporation of ganglionic eminences of rat’s E16 brain slices indicating an increased migration of interneurons when neuritin is over-expressed. Post-translational modifications of alpha-tubulin are known to be associated with microtubule dynamics. ICC and Western blot analyses showed an enrichment of stable microtubules, that likely reflects the diminished migratory capability of the cells, in neuritin silenced cells; moreover, neuritin over-expression, that induces an increase in the migratory ability in the cells, is also able to up-regulates the tyrosinated form of a-tubulin reflecting the presence of more dynamic microtubules. Altogether these data demonstrate a novel function of neuritin in promoting migration of neuronal cells, both in vitro and in vivo, through the modulation of microtubule stability, and makes neuritin a good candidate as a therapeutic target for different diseases in which cellular migration is involved. Cappelletti, G., M. Galbiati, et al. (2007). "Neuritin (cpg15) enhances the differentiating effect of NGF on neuronal PC12 cells." J Neurosci Res 85(12): 2702-2713. Cariboni, A., R. Maggi, et al. (2007). "From nose to fertility: the long migratory journey of gonadotropin-releasing hormone neurons." Trends Neurosci 30(12): 638-644. Maggi, R., F. Pimpinelli, et al. (2000). "Immortalized luteinizing hormone-releasing hormone neurons show a different migratory activity in vitro." Endocrinology 141(6): 2105-2112. Nedivi, E., D. Hevroni, et al. (1993). "Numerous candidate plasticity-related genes revealed by differential cDNA cloning." Nature 363(6431): 718-722. Putz, U., C. Harwell, et al. (2005). "Soluble CPG15 expressed during early development rescues cortical progenitors from apoptosis." Nat Neurosci 8(3): 322-331. Radovick, S., S. Wray, et al. (1991). "Migratory arrest of gonadotropin-releasing hormone neurons in transgenic mice." Proc Natl Acad Sci U S A 88(8): 3402-3406.

The nutritional support in orthopedic surgery is an unclear idea to date. In these years, the goal of this project has been to clarify what is meant by nutritional support in patients undergoing hip or knee replacement and spine surgery, demonstrating its value in relation to the perioperative path. This aim was pursued by applying a methodology of first studying the literature, then analyzing the setting, evidencing shortcomings, proposing management protocols, and verifying the effectiveness of nutritional strategies. The literature search highlighted the multiple phases that should be coordinated, including prehabilitation, perioperative, and postdischarge management. When applying optimization strategies, such as anemia correction, it may be necessary to examine the eating behavior and anticipate the metabolic responses according to the patient’s state of health. The predictive potential of hemoglobin on the clinical outcome in spine surgery has been investigated through retrospective analysis of existing data, suggesting the need to refine decision-making algorithms that identify the patients to be treated, underlining the importance of having high hemoglobin values in the preoperative period to have more chances of clinical success. Prospective observational studies confirmed that patients either after spine surgery or arthroplasty do not meet their needs for what concerns energy and proteins. It seems to be vital to redefine the tactics to promote early oral nutrition, counteract the depletion of body reserves, and acknowledge the impairment of the olfactory stimulus caused by anesthetic drugs. More transparent information on diet and nutrition should be conveyed to patients. The nutritional support program might include a healthy eating education that teaches the concepts of nutritional value, quantity, quality, and timing. Clear information on what to eat or not to eat before surgery must be accompanied by dietary advice also for postoperative recovery. New management proposals can integrate aspects of healthy eating education into a multimodal approach that includes advice on increasing physical activity and promoting night sleep. A risk management system inspired by the hazard analysis and critical control points system can be effectively applied before orthopedic surgery to manage malnutrition. The interventional trials proved that nutrient deficiencies, including those of vitamin D and iron, are common in the surgical population. Dietary supplements should be carefully selected for what concerns the pharmaceutical formula, as well as for the content of important cofactors that may be necessary to boost the metabolic response. Early cardiovascular effects can derive from correcting hypovitaminosis D with a liquid form of vitamin D, and promising data on enhancing preoperative hemoglobin values can derive from using a supplement containing iron and B vitamins. The future of perioperative medicine in orthopedic surgery should choose a pragmatic integration of dietary and nutritional aspects in orthopedics, thus possibly improving health performance and patient experience.

The major mechanism for the repair of DNA doublestrand breaks (DSBs) in mammalian cells is non-homologous end-joining (NHEJ), a process that involves the DNA-dependent protein kinase , XRCC4 and DNA ligase IV. Rodent cells and mice defective in these components are radiation-sensitive and defective in V(D)J-recombination, showing that NHEJ also functions to rejoin DSBs introduced during lymphocyte development. We have generated a knock-in mouse model with a homozygous Lig4 arginine to histidine (R278H) mutation that corresponds to the mutation identified in the first LIG4-deficient patient, who developed T cell leukemia associated with increased cellular radiosensitivity. The clinical presentation of the syndrome is complex and heterogeneous and may include varying degrees of lymphopenia, growth retardation and microcephaly. The phenotypic effects of the impaired repair of non programmed DNA damage are more diverse and difficult to study. Although such defects in cell survival and proliferation are likely to have an impact on the immune system, their contribution to the immunodeficiency of the LigIV syndrome remains unknown.

Thyroid cancer is the most common endocrine malignancy and his global incidence has rapidly increased in last decades. Despite the major part of thyroid cancer is represented by well differentiated hystotypes, the acquisition of additional mutations, such as p53 and β-catenin ones, causes loss of differentiation and confer high malignancy. Current treatment for undifferentiated thyroid cancers is regarded as almost ineffective, with a median survival of 3- 4 months, somewhat better in localized and worse in metastatic disease. SP600125 is a multi-kinase inhibitor that has recently been shown to be a promising anticancer drug. In the last five years it has been proved able to induce endoreduplication and subsequent polyploidization, but there are contrasting results about its intracellular actions and there is no evidence on the specific mechanism of action. Moreover, in 2012, SP600125 has been found to be the most effective against p53 deficient cells among more than 300 screened compounds. However, opposite results have also been obtained depending on cell type, concentration and time of incubation. In the current study the effects of micromolar doses of SP600125 have been characterized in six thyroid cancer cell lines with different p53 status. The results show that at low concentrations SP600125 dramatically reduces the proliferation of p53 mutated cells, with lesser effects on p53 null and no effects on the wild-type ones. In p53 mutated cells it has been proved able to induce p53 nuclear translocation and phosphorylation at serine 15; this modification resulted to be responsible of increased levels of p21. Importantly other considerable novel effects have been revealed. Firstly, SP600125 caused alterations of microtubule dynamics in p53 mutated cells, with increase of acetylation levels and loss of Microtubule Organizing Center (MTOC)-periphery organization. These effects were accompanied by alterations in cellular morphology and in late endosome/lysosome trafficking. Endoreduplication and alteration of microtubule dynamics finally resulted in aberrant mitosis and cell death. The second mechanism involves alteration of cellular motility: different kinases involved in this process are affected by SP600125 treatment and β-catenin remains at the intercellular junctions in affected cells,consistent with a failure of cell detachment (a figure consistent with inhibition of cell migration/motility). Microtubule alterations concomitantly also account for motility alterations. Both tubulin and β-catenin variations are due to HDAC6 activity alterations. This enzyme regulates the levels of acetylation of these proteins and is profoundly inhibited following SP600125 in p53 deficient cells. Alteration of HDAC6 activity is hypothesized to be the result of SP600125 direct inhibition of ROCK2, an upstream kinase regulator of HDAC6 activity. In conclusion SP600125 is a promising drug particularly active on p53-mutated cancers at concentrations unable to affect normal cell viability. The effects of SP600125 action include arrest of tumor growth, and importantly induction of tumor cell death and metastatic diffusion inhibition.

Introduction Epithelial homeostasis is guaranted by somatic stem cells wich, through different patways, such as p63 and Notch signaling, self-renew, differentiate and control tissue function and integrity. In vivo behavior of epithelial stem cells (ESCs) from different epithelia reflects differences in physiological role of the tissue. Limbal stem cells deficiency (LSCD) is characterized by conjunctival epithelial ingrowth, neovascularization, recurrent corneal erosion and persistent ulcers, as well as corneal scarring and ultimately leads to visual impairment and blindness. When the cornea is entirely covered with a fibro-vascular tissue, the chances of success of a traditional penetrating keratoplasty are virtually absent. Transplantation of autologous limbal epithelial stem cells (LESCs) or oral mucosa epithelial stem cells (OMESCs) cultured on fibrin glue has shown to be successful for unilateral or bilateral LSCD treatment, respectively. Despite that, fibrin presented several limitations including inability to repair or replace damaged corneal stroma. Aim The purpose of this work is to better understand the potentiality of somatic epithelial stem cells in order to identify possible approaches to exploit this potentiality. In the light of this, my research group has been able to identify and set up a cell therapy approach for a unique homozygous-heterozygous mosaicism of EEC Syndrome, demonstrating that epithelial stem cells have an intrinsic potential for regenerative medicine that can be exploited with a deeper characterization. We, also, aimed to test human keratoplasty lenticules (HKLs): particularly attractive, full-thickness scaffolds for corneal epithelial and stromal reconstruction that provides an interesting organotypic culture system for evaluation of growth, proliferation, and differentiation processes of epithelial stem cells (ESCs). Results During the three years of PhD I had the opportunity to collect and review cellular biology data of four diverse types of primary epithelial cells, derived from four different epithelia (skin, oral mucosa, limbus/cornea and conjunctiva) and, to better investigate the exhaustion of clonogenic potential and the self-renewal of epithelial stem cells we took in consideration also p63-defective oral mucosa primary cell lines obtained from three patients affected by EEC syndrome, already known to cause an acceleration in epithelial aging. Demonstrating that epithelial stem cells have an intrinsic potential for regenerative medicine that can be exploited with a deeper characterization, we have also been able to apply a cell therapy protocol to a patient affected by EEC syndrome in a rare form of mosaicism. We isolated the cell population with a mild phenotype from this patient, enriching these primary cells in vitro and producing well-organized and stratified epithelial sheets. The novelty and the importance of this case was related to the possibility to start a customized cell therapy approach for this unique case of EEC syndrome, based solely on epithelial stem cell manipulation. Limbal stem cells (H-LESCs) expanded onto HKLs gave rise to a keratinized stratified squamous epithelium morphologically similar to that of normal corneas. To set up the cohort of animal patients, we proceded with the characterization of cell lines obtained from biopsies of the cornea (C-CESCs) and oral mucosa (C-OMESCs) of canine origin. Primary lines were serially propagated until exhaustion in order to get life span data to compare their behavior with human limbal stem cells. For each passage, we also performed colony forming efficiency assays (CFE) to estimate the proportion of clonogenic cells present in the culture. Results showed a trend of canine cell lines comparable to human limbal cells trend, with a similar decrease of clonogenic cells number, a similar percentage of aborted colonies during serial cultivation and a similar replicative senescence. Then, primary human and canine epithelial stem cells were seeded onto HKLs. The resulting epithelia was well organized and stratified into four to five cell layers with basal cuboidal cells differentiating upward to winged cells. The layer of basal cuboidal cells was firmly attached to the underlying ECM and to the basement membrane through integrinβ4. Maintenance of stemness potential and differentiation pathways were assessed checking the expression of the stem cells marker p63 and the terminally differentiated cells marker Involucrine. Importantly, expression of the different markers resembled that observed in normal epithelia, thus suggesting that HKLs are able to support the growth and maintain the differentiation pathways of epithelial stem cells. Discussion and conclusions Our findings demonstrate that primary epithelial cells have unique characteristics with an inimitable potential that makes them a malleable tool, able to adapt to different necessities. And so on, although these data are intriguing, further investigation could provide more and more useful data for their clinical application in regenerative medicine. As a proof-of-principle that epithelial stem cells have an intrinsic potential for regenerative medicine that can be exploited with a deeper characterization, my group have been able to apply, in vitro, a cell therapy protocol to a patient affected by a rare mosaic form of EEC syndrome. HKLs appear to be particularly attractive, animal-free (feeder-free) and full-thickness scaffolds for corneal reconstruction. We have already started with the the recruitment of canine patients to assess the transplantability and functionality of these organotypic structures and, through the collaboration with different veterinary departments, we are creating a small cohort on which to start with first transplantation trials.
Introduzione L'omeostasi epiteliale è garantita da cellule staminali somatiche che, attraverso diversi pathways, come quello di p63 o di Notch, si auto-rinnovano, differenziano e controllano la funzione e l'integrità del tessuto. Il comportamento in vivo delle cellule staminali epiteliali (ESC) di diversi epiteli riflette le differenze nel ruolo fisiologico che queste cellule hanno nel tessuto di origine. Il deficit di cellule staminali limbali (LSCD) è caratterizzato da crescita epiteliale congiuntivale, neovascolarizzazione, erosione corneale ricorrente e ulcere persistenti, nonché cicatrici corneali, e porta infine a deficit visivo e cecità. Quando la cornea è interamente coperta da un tessuto fibro-vascolare, le probabilità di successo di una cheratoplastica penetrante tradizionale sono praticamente assenti. Il trapianto di cellule staminali epiteliali autologhe del limbus (LESCs) o di cellule staminali epiteliali della mucosa orale (OMESCs) coltivate su scaffold di fibrina ha dimostrato di avere successo per il trattamento unilaterale o bilaterale di LSCD, rispettivamente. Nonostante ciò, la fibrina presentava numerose limitazioni tra cui l'incapacità di riparare o sostituire lo stroma corneale danneggiato. Scopo Lo scopo di questo lavoro è quello di comprendere meglio la potenzialità delle cellule staminali epiteliali somatiche al fine di identificare possibili approcci per sfruttare questa potenzialità. Alla luce di ciò, il mio gruppo di ricerca è stato in grado di identificare e impostare un approccio di terapia cellulare per un caso unico di mosaicismo omozigote-eterozigote della Sindrome EEC, dimostrando che le cellule staminali epiteliali hanno un potenziale intrinseco per la medicina rigenerativa che può essere sfruttata con una caratterizzazione più profonda. Un secondo scopo della tesi è stato quello di testare gli Human Keratoplasty Lenticules (HKL): scaffolds particolarmente attraenti per la ricostruzione a tutto spessore della superficie anteriore dell’occhio che fornisce, anche, un interessante sistema di coltura organotipica per la valutazione dei processi di crescita, proliferazione e differenziazione delle cellule staminali epiteliali (ESCs ). Risultati Durante i tre anni di dottorato ho avuto l'opportunità di raccogliere e rivedere i dati di biologia cellulare di quattro diversi tipi di cellule epiteliali primarie, derivate da quattro diversi epiteli (pelle, mucosa orale, limbus/cornea e congiuntiva) e, per studiare meglio l'esaurimento del potenziale clonogenico e dell'auto-rinnovamento delle cellule staminali epiteliali abbiamo preso in considerazione anche delle linee cellulari primarie di mucosa orale con difetti genetici del gene p63 già noti per causare un'accelerazione nell'invecchiamento epiteliale, ottenute da tre pazienti affetti da sindrome EEC. Dimostrando che le cellule staminali epiteliali hanno un potenziale intrinseco per la medicina rigenerativa che può essere sfruttato con una caratterizzazione più profonda, siamo stati anche in grado di applicare un protocollo di terapia cellulare a un paziente affetto da sindrome EEC in una rara forma di mosaicismo. Abbiamo isolato la popolazione cellulare con un fenotipo lieve da questo paziente, arricchendo queste cellule primarie in vitro e producendo foglietti epiteliali ben organizzati e stratificati. La novità e l'importanza di questo caso è legata alla possibilità di iniziare un approccio di terapia cellulare personalizzata per questo caso unico di sindrome EEC, basata esclusivamente sulla manipolazione delle cellule staminali epiteliali. Le cellule staminali limbari (H-LESC) coltivate su HKL hanno dato origine a un epitelio squamoso stratificato cheratinizzato morfologicamente simile a quello delle cornee normali. Per costituire la coorte di pazienti animali, abbiamo proceduto alla caratterizzazione di linee cellulari ottenute da biopsie della cornea (C-CESCs) e mucosa orale (C-OMESCs) di origine canina. Le linee primarie sono state propagate in serie fino ad esaurimento al fine di ottenere dati di life span per confrontare il loro comportamento con le cellule staminali limbari umane. Per ogni passaggio, abbiamo anche eseguito analisi di Colony Forming Efficiency (CFE) per stimare la proporzione di cellule clonogeniche presenti nella coltura. I risultati hanno mostrato una tendenza delle linee cellulari canine comparabile all'andamento delle cellule limbari umane, con una diminuzione simile del numero di cellule clonogeniche, una percentuale simile di colonie abortive durante i passaggi in coltura e una simile senescenza replicativa. Quindi, cellule staminali epiteliali primarie umane e canine sono state seminate su HKL. L'epitelio risultante era ben organizzato e stratificato in quattro o cinque strati cellulari con cellule cuboidali basali che differenziano le cellule verso l'alto e quelle alate. Lo strato di cellule cuboidali basali era saldamente attaccato alla matrice extracellulare sottostante e alla membrana basale attraverso l'integrina-4. Sono stati valutati il mantenimento del pool di cellule staminali e i corretti processi di differenziamento controllando l'espressione del marcatore di cellule staminali, p63, e del marcatore di cellule terminalmente differenziate, Involucrina. È importante sottolineare che l'espressione dei diversi marcatori assomiglia a quella osservata negli epiteli normali, suggerendo quindi che gli HKL sono in grado di supportare la crescita e il mantenimento di tessuti epiteliali ricostruiti. Discussione e Conclusioni I nostri risultati dimostrano che le cellule epiteliali primarie hanno caratteristiche uniche con un potenziale inimitabile che le rende uno strumento malleabile, in grado di adattarsi alle diverse necessità. Sebbene questi dati siano intriganti, ulteriori indagini potrebbero fornire dati sempre più utili per la loro applicazione clinica nella medicina rigenerativa. Come prova di principio che le cellule staminali epiteliali hanno un potenziale intrinseco per la medicina rigenerativa che può essere sfruttato con una caratterizzazione più profonda, il mio gruppo è stato in grado di applicare, in vitro, un protocollo di terapia cellulare a un paziente affetto da un raro mosaicismo di sindrome EEC. Le HKL sembrano essere scaffold particolarmente attraenti (animal-free) per la ricostruzione a tutto spessore della cornea. Abbiamo già iniziato con il reclutamento di pazienti canini per valutare la trapiantabilità e la funzionalità di queste strutture organotipiche e, attraverso la collaborazione con diversi dipartimenti veterinari, stiamo creando una piccola coorte sulla quale iniziare con le prime prove di trapianto.

GnRH analogues are used for the treatment of prostate cancer (PCa) because their ability to suppress the activity of the pituitary-testicular axis, with consequent blockade of testosterone production. However, after an initial responsiveness to hormonal deprivation, PCa progresses and then metastatises. It is known that the system of the plasminogen activator (uPA, uPA inhibitors PAI-1/2 and uPA receptor, uPAR) has been involved in the local degradation of the extracellular matrix and PCa progression and metastases. Studies performed in our laboratory have demonstrated the presence of GnRH receptors, suggesting a direct effect of GnRH analogues in inhibiting the proliferation of human PCa cell lines. The aim of this study was to test the effect of an agonist (Leuprolide) and an antagonist (Cetrorelix) of GnRH on uPA/uPAR and PAI-1 expression and activity, on the migratory and invasion capabilities in the two androgen-independent cell lines, DU145 and PC3 cells. The results obtained in DU145 and PC3 cells show that both Leuprolide and Cetrorelix: 1) significantly decrease the enzymatic activity of uPA; 2) induce a marked decrease of uPA and a significant increase of PAI-1 protein levels; 3) increase the presence of soluble uPAR in the cell media; 4) decrease the migratory and invasion capabilities. In conclusions, GnRH analogues might interfere with the mechanisms of metastatic progression of human androgen-independent PCa by inhibiting the activity of the plasminogen activator system.

The hepatocyte growth factor (HGF)/Met receptor signaling pathway is deregulated in diverse human malignancies and plays a central role in oncogenesis, tumor progression and invasive cancer cell growth. Additionally, altered expression and splicing (i.e. inclusion of variable exon-5 (v5)) of the cell adhesion marker, CD44, is associated with advanced cancer phenotypes. We sought to understand how HGF regulates CD44v5 expression. The immortalized non-tumorigenic keratinocyte (HaCaT) cell line, abundantly expresses both the tyrosine kinase growth factor receptor, c-Met, and the transmembrane glycoprotein CD44v5. HGF stimulation increased HaCaT cell migration and CD44v5 protein expression. HGF-dependent CD44v5 up-regulation required activation of the ERK1/2 MAPK module and phosphorylation of Sam68, a protein involved in RNA processing, splicing, and CD44 variant 5 inclusion. Knock-down of Sam68 or treatment of cells with the MEK inhibitor (U0126) blocked CD44v5 expression and cell migration in response to HGF. Similar to HaCaT cells, highly migratory MDA-MB-231 breast cancer cells also required Sam68 expression for HGF-induced migration. However, in these cells, HGF-induced migration occurred independently of ERK1/2 activation or CD44v5 (or v6) expression, but instead required ERK5 signaling. Phospho-mutant, but not wt-Sam68, blocked HGF-induced cell migration in both cell types. These results suggest that Sam68 acts as a convergence point for ERK-dependent inputs governing the expression of cell-specific gene targets. Future experiments will be aimed at elucidation of Sam68-regulated (splice variants) genes required for HGF-induced breast cancer cell migration. Blockade of Sam68, a key mediator of cell migration, may provide a new avenue for therapeutic inhibition of metastatic cancers.

Three-dimensional (3D) cancer models are overlooking the scientific landscape with the primary goal of bridging the gaps between two-dimensional (2D) cell cultures, animal models and clinical research. In this thesis, we describe an innovative tissue engineering approach applied to colorectal cancer (CRC) starting from decellularized human biopsies in order to generate an organotypic 3D bioactive model. This in vitro 3D system recapitulates the ultrastructural environment of native tissue as demonstrated by histology, immunohistochemistry, immunofluorescence and scanning electron microscopy analyses. Mass spectrometry of proteome and secretome confirmed a different stromal composition between decellularized healthy mucosa and CRC in terms of structural proteins (COL1A1, COL1A2, and COL3A1) and secreted proteins such as DEFA3. Importantly, we proved that our 3D acellular matrices retained their biological properties: using CAM assay, we observed a decreased angiogenic potential in decellularized CRC compared with healthy colon mucosa, caused by direct effect of DEFA3. In addition, we demonstrated that following a 5 days of recellularization with HT-29 cell line, the 3D tumor matrices induced an over-expression of IL-8, a DEFA3-mediated pathway and a mandatory chemokine in cancer growth and proliferation, compared with recellularized healthy mucosa and 2D conventional culture model. Given the biological activity maintained by the scaffolds after decellularization, we believe this approach is a powerful tool for future pre-clinical research and screenings.
I modelli tumorali tridimensionali (3D) si stanno affacciando sul panorama scientifico con l’obiettivo primario di superare le limitazioni di colture cellulari convenzionali (2D) e modelli animali negli approcci di ricerca clinica. In questa tesi di dottorato, si descrive un innovativo approccio di ingegneria tissutale applicata alla ricerca oncologica mediante il quale, partendo da una biopsia tissutale decellularizzata, si genera un modello organo-tipico 3D bioattivo. Questo modello 3D, ricapitola, in vitro, l’ambiente ultra-strutturale del tessuto nativo come dimostrato da indagini istologiche, immunoistochimiche, di immunofluorescenza e di microscopia elettronica a scansione. L’analisi del proteoma e del secretoma mediante spettrometria di massa ha confermato una differente composizione stromale tra la mucosa colica sana decellularizzata e quella della controparte tumorale (CRC) in termini di proteine strutturali (Collagene 1A1, Collagene 1A2, Collagene 3A1) e di proteine secrete, come la Defensina alfa 3. Abbiamo dimostrato che le nostre matrici 3D mantengono le loro proprietà biologiche dopo il processo di decellularizzazione: mediante la CAM, abbiamo osservato un decremento del potenziale angiogenico della matrice decellularizzata di CRC comparata con la mucosa colica sana, causata da un effetto diretto della Defensina alfa 3. Inoltre, abbiamo dimostrato che dopo 5 giorni di ricellularizzazione con cellule HT-29 (linea stabilizzata di cancro del colon), le matrici tumorali 3D (comparate con le rispettive mucose coliche sane ed il metodo di coltura 2D) hanno indotto una sovra-espressione di IL-8, una chemochina a valle del pathway della Defensina alfa 3, che gioca un ruolo molto importante nella crescita e proliferazione tumorale. In conclusione, avendo dimostrato la capacità dei delle nostre matrici acellulari 3D di mucosa colica sana e CRC di mimare gli stimoli ultra-strutturali e biologici dei rispettivi tessuti nativi, crediamo che questo approccio possa essere un efficace strumento per migliorare il livello delle ricerche precliniche e nei test di screening di farmaci.

There is a growing interest by scientific community, together with pharmaceutical companies and clinical researcher on finding valid alternatives to standard models in the development of new therapeutic strategies and robust drug screening processes. Research still relies on the use of immortalized cell lines, primary cell extracted from living organs and animal models. These methodologies are valid and still mainstream for general purposes, but most of the times present evident limits. Cell lines and primary cells often fail to reproduce the exact characteristics of the tissue found in vivo, because they lose some of the features and functionalities when grown in culture. On the other hand, animal models, even if necessary for the study of specific diseases and new compound testing, are expensive and time-consuming, and often show poor predictive capacity and scarce reproducibility of the human condition. Hence, human pluripotent stem cells could represent a valid alternative to the existing models, thank to their capacity to be expanded indefinitely and differentiate into almost all cell types found in vivo. In the recent years, there has been a growing attention on engineered tissues differentiate from pluripotent cells. They have the capacity to transform the way to study human pathophysiology and physiology in vitro. Nevertheless, there are still major problems in the process of differentiating human embryonic and induced pluripotent stem cell in vitro. This is because of the difficulty to specifically direct cell fate to a particular cell type in a robust way, and for the poor reproduction of the physiological conditions under which these processes take place. In this direction, new microtechnologies could help overcome these major limitations, because they allow working on microscales, in a way that cannot be reproduced by standard culture conditions. In this context, the aim of this PhD thesis is to efficiently differentiate human pluripotent stem cells in order to obtain functional relevant cell types, such as cardiomyocytes and hepatocytes. The strategy applied for the obtainment of these human in vitro models rely on the application of microscale technologies for reproducing in vitro the main physiological cues, which guide differentiation and allow functional development. In particular, three-dimensional microwell were used for modulating endogenous factor accumulation on differentiating human embryoid bodies, to screen for selective germ layer commitment guided by a differential microenvironment around the differentiating cells. Furthermore, a mechanical modulation of the pluripotent nuclei mechanical properties was imposed with the use of microstructured substrate, for the study of the peculiar capacity of nuclear cell deformation, and its effect in pluripotency and early differentiation. Moreover, microfluidic technologies were used to selectively modulate cell soluble microenvironment, in order to optimize pluripotency maintenance, early germ layer commitment, and functional differentiation into cardiomyocytes and hepatocytes. A high percentage of spontaneously beating cardiomyocytes on chip was obtained, showing proper functional response to calcium stimuli. On the other hand, microfluidic technology allowed to obtain a higher percentage of hepatocytes compared to standard culture conditions. These cells showed proper phenotypic and functional characteristics, which were also analyzed in a more physiological condition under a defined oxygen gradient, mimicking in vivo physiological conditions. These specific cell types generated on chip from human pluripotent stem cells through a multi-stage approach show specific functional differentiation, which opens a new perspective for multi-parametric and large scale human tissue-based screening assays.
Vi è un crescente interesse fra la comunità scientifica internazionale, le compagnie farmaceutiche e la ricerca clinica nel trovare delle valide alternative ai modelli standard nello sviluppo di nuove strategie terapeutiche e di processi di scoperta di nuovi farmaci. I ricercatori si basano tuttora sull’utilizzo di linee cellulari immortalizzate, cellule primarie estratte da organi e su modelli animali. Queste metodiche sono valide e largamente utilizzate per scopi generali, ma il più delle volte presentano dei limiti evidenti. Le linee cellulari e cellule primarie spesso non riproducono fedelmente le esatte caratteristiche dei tessuti in vivo, poiché perdono alcune caratteristiche fisiche e funzionali quando sono coltivate in vitro. D’altra parte, i modelli animali sono ancora necessari per lo studio di patologie specifiche e nel test di nuovi composti, ma risultano essere dispendiosi in termini di tempo e denaro e spesso mostrano una scarsa capacità predittiva nei confronti degli effetti sull’uomo. Quindi, le cellule staminali pluripotenti umane rappresentano una valida alternativa ai modelli esistenti, grazie alle loro capacità di essere espanse in vitro indefinitamente e di poter differenziare in tutti i tipi cellulari derivanti dai tre foglietti germinali. Negli ultimi anni l’attenzione si è concentrata sui tessuti ingegnerizzati, differenziati a partire da cellule pluripotenti. Questi hanno la possibilità di trasformare radicalmente il modo in cui studiamo in vitro la fisiologia e la patofisiologia umana. Ciò non di meno, vi sono ancora problemi nei processi di differenziamento di cellule staminali embrionali e pluripotenti indotte umane in vitro. Questo perché si riscontra difficoltà nel dirigere specificamente il destino cellulare verso un determinato tipo cellulare in modo robusto, e per la scarsa riproducibilità delle condizioni fisiologiche in cui questi processi hanno normalmente luogo in vivo. In questo ambito, le nuove micro-tecnologie possono dare un aiuto nell’oltrepassare queste limitazioni, poiché permettono di lavorare in micro-scala in maniera difficilmente riproducibile in condizioni di coltura standard. In questo contesto, lo scopo di questa tesi di dottorato è quello di differenziare efficacemente cellule staminali pluripotenti umane per riuscire ad ottenere rilevanti tipi cellulari, quali cardiomiociti ed epatociti. La strategia applicata per l’ottenimento di questi modelli umani in vitro si basa sull’applicazione di tecnologie in micro-scala per permettere la riproduzione in vitro delle nicchie fisiologiche, che guidano il differenziamento e permettono lo sviluppo funzionale. In particolare sono stati utilizzati dei micro-pozzetti tridimensionali per modulare l’accumulo di fattori endogeni in corpi embrioidi umani in differenziamento, per studiare lo sviluppo dei tre foglietti germinali guidato da diversi microambienti cellulari. È stata poi imposta una modulazione delle proprietà meccaniche dei nuclei di cellule pluripotenti tramite utilizzo di substrati micro-strutturati, per lo studio della capacità peculiare di deformazione nucleare di tali cellule e si è valutato l’effetto sulla pluripotenza e il differenziamento precoce. Inoltre sono state utilizzate tecnologie micro-fluidiche per modulare selettivamente il microambiente cellulare solubile, in modo da ottimizzare il mantenimento della pluripotenza in chip micro-fluidici, nonché lo sviluppo cellulare precoce e il differenziamento funzionale in cardiomiociti ed epatociti. È stata ottenuta un’alta percentuale di cardiomiociti contrattili nei chip che mostravano risposte funzionali corrette a stimoli di calcio. La tecnologia micro-fluidica ha permesso poi di ottenere un’alta percentuale di epatociti in chip rispetto alle condizioni di coltura standard. Queste cellule mostravano caratteristiche fenotipiche e funzionali corrette, che sono state poi analizzate in condizioni più fisiologiche sotto un gradiente stabile di ossigeno, mimando le condizioni in vivo. Questi tipi cellulari specifici, generati in chip da cellule staminali pluripotenti umane tramite un approccio multi-stadio, mostrano un differenziamento funzionale specifico, che apre a nuove prospettive per test multi-parametrici su larga scala basati su tessuti funzionali umani.

Transformed cells undergo profound reprogramming of cellular metabolism to ensure high rate of proliferation (Hanahan D, 2011). Mitochondria are important for both bioenergetics and biosynthetic pathways, which have to be coordinated. Different cancer types heterogeneously express mitochondrial genes likely reflecting the existence of different mitochondrial pathways providing adaptation to altered needs of cancer cell metabolism. In this work we explored this hypothesis in breast cancer. Breast cancer is heterogeneous disease classified according to diverse clinical and pathological features, histochemical markers and oncogenic transcriptional programs, that identify five breast cancer subtypes (basal-like, Her2-enriched, Luminal A, Luminal B and normal-like) (Sørlie T et al., 2001; Perou CM et al., 2000). So far, the mitochondrial transcriptional patterns were not considered as a basis of classification. In this work we define for the first time breast cancer Mitochondrial Tumour Subtypes (MTSs), upper fork (UF) and lower fork (LF), according to their nuclear-encoded mitochondrial transcription profile. UF and LF are characterised by two groups of mitochondrial genes co-regulated in opposite way. The classification was performed applying the MCbiclust algorithm (Bentham et al., 2016, preprint) to a mitochondrial gene set (MitoCarta, Pagliarini et al., 2008). The experimental characterisation of UF and LF revealed distinct bioenergetics and metabolic features. Interestingly, in basal condition UF has higher mitochondrial content, characterised by higher expression of CI and CIV resulting in higher respiration rate. At cellular level metabolism, UF and LF revealed different arrangement of metabolic enzymes belonging to particular pathways of the intermediate metabolism, UF cells express enzymes of which substrates are glucose-derived, whereas LF cells preferentially use enzymes exploiting glutamine-derived substrates. Moreover, metabolic flux analysis confirmed the substrate preference driven by the MTSs to feed into the TCA cycle, in particular UF cells mitochondria prefer glucose derived pyruvate while LF mitochondria catabolise glutamine. In addition, experiments in restricted nutrient conditions were carried out. These further confirmed which is the substrate preferentially used by MTSs to sustain cell functionality. Moreover, the same experiments also revealed the activation of different mitochondrial biogenesis programs during deprivation treatments, both looking at mitochondrial protein expression and mtDNA content. Overall, the mitochondrial biogenesis pattern associated with the MTSs drives nutrient choice and metabolic program, leading to a definition of the concept of substrate preference, new insight in the field of reprogramming of bioenergetics and cancer metabolism.
Le cellule tumorali vanno incontro a profonde modifiche del proprio metabolismo cellulare per assicurarsi un alto tasso di proliferazione (Hanahan D, 2011). I mitocondri sono una sede importante sia per la produzione di energia che per i processi biosintetici, pertanto queste vie devono essere opportunamente coordinate. Molti tipi di cancro esprimono geni mitocondriali in modo eterogeneo, questo probabilmente riflette l’esistenza di diverse vie mitocondriali in grado di fornire diverse capacità di adattamento ad un metabolismo alterato, come lo è il profilo metabolico associato ai tumori. In questo lavoro abbiamo esplorato questa ipotesi nel modello tumorale del cancro alla mammella. Il cancro alla mammella rappresenta una malattia comunemente classificata sulla base di caratteristiche cliniche e patologiche, marcatori istochimici e programmi di trascrizione genica che identificano cinque sottotipi di tumore alla mammella (basal-like, Her2-enriched, Luminal A, Luminal B e normal-like) (Sørlie T et al., 2001; Perou CM et al., 2000). Tuttavia, l’espressione di geni mitocondriali non è stata finora considerata nella classificazione di questi tumori. In questo lavoro definiamo per la prima volta sottotipi tumorali mitocondriali di cancro alla mammella (MTSs), upper fork (UF) e lower fork (LF), definiti sulla base del loro profilo di trascrizione di geni mitocondriali di codificazione nucleare regolati in modo opposto. La classificazione è stata fatta utilizzando l’algoritmo MCbiclust (Bentham et al., 2016, preprint) su un set di geni mitocondriali (MitoCarta, Pagliarini et al., 2008). Dalla caratterizzazione sperimentale di UF e LF sono emerse caratteristiche bioenergetiche e metaboliche distintive. In particolare, in condizioni basali UF ha un maggiore contenuto mitocondriale, caratterizzato da una maggiore espressione dei complessi I e IV della catena respiratoria, che culmina in un tasso di respirazione maggiore. A livello di metabolismo cellulare, UF e LF mostrano un diverso assetto di enzimi metabolici appartenenti a particolari vie del metabolismo intermedio. In particolare le cellule dell’UF esprimono enzimi i cui substrati sono derivati dal glucosio, mentre le cellule di LF preferiscono utilizzare enzimi i cui substrati sono derivati dalla glutammina. Inoltre l’analisi dei flussi metabolici ha confermato la preferenza di substrato determinata dai MTSs per alimentare il ciclo di Krebs. In particolare i mitocondri delle cellule dell’UF preferiscono il piruvato derivato dal glucosio, mentre i mitocondri di LF catabolizzano la glutammina. In aggiunta, sono stati eseguiti esperimenti con concentrazioni di nutrienti ridotte, i quali, in accordo con i risultati precedenti, hanno ulteriormente confermato i substrati preferenzialmente utilizzati dai MTSs per sostenere la funzionalità cellulare. Inoltre gli stessi esperimenti hanno rivelato l’attivazione di diversi programmi di biogenesi mitocondriale indotti dai trattamenti, sia a livello di espressine di proteine mitocondriali che in termini di contenuto di DNA mitocondriale. Complessivamente, il pattern di biogenesi mitocondriale associato ai sottotipi tumorali mitocondriali determina la scelta di nutrienti e programmi metabolici, portando così alla definizione del concetto di preferenza di substrato, il quale fornisce una nuova visione nel campo della riprogrammazione bioenergetica e metabolica del cancro e la possibilità di classificare sottotipi tumorali sulla base dell’espressione dei geni mitocondriali.

ABSTRACT Background: Park2 mutations cause Autosomal Recessive Juvenile Parkinsonism (ARJP), characterized by the loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc). Park2 encodes for a ubiquitin-ligase protein whose mutation upregulates Gluk2, a subunit of the glutamate kainate receptor (KAR), expressed in SNpc neurons. Park2 is highly expressed also in astrocytes and KARs upregulation may induce excitotoxicity both in DA neurons and glia, leading to an early synaptopathy, neuroinflamation and neurodegeneration. Aims and Objectives: 1. To generate Park2 induced pluripotent stem cells (iPSCs)-derived in vitro cellular models; 2. To characterize Park2 iPSCs-derived in vitro cellular models; 3. to test glutamate toxicity due to KAR upregulation in Park2 cellular models. Materials and Methods: Fibroblasts and lymphocytes from Park2 patients and age-matched controls were reprogrammed into iPSCs. The iPSCs were further differentiated into dopaminergic neurons, astrocytes and mesencephalic organoids were generated and differentiated. Protein expression profile was analysed through western blot (WB), qPCR and immunofluorescence (IF). Electrophysiology assessment was performed on dopaminergic neurons and midbrain organoids in order to better functionally profile these models. Results: Gluk2 levels resulted significantly increased in PARK2 midbrain organoids compared to CTR both at WB (p< 0.001) and qPCR analyses (p< 0.001). Gluk2 levels resulted also significantly enhanced in PARK2 astrocytes both at WB (p< 0.05) and qPCR analyses (p< 0.05). TH mRNA and protein levels were significantly increased both in PARK2 dopaminergic neurons (WB p< 0.01; qPCR p< 0.0001; IF p< 0.0001) and midbrain organoids (WB p< 0.01; qPCR p< 0.0001; IF p< 0.0001) compared to CTR. Glial fibrillary acidic protein (GFAP), a marker of reactive astrocytes, resulted enhanced in PARK2 astrocytes and especially in PARK2 midbrain organoids (WB p< 0.001; IF p< 0.01). EAAT2, the astrocytic glutamate transporter resulted reduced in mutated lines (WB p< 0.01). Calcium-imaging and HD-MEAs show an oscillatory augmented reactivity in PARK2 midbrain organoids. Conclusions and perspectives: Gluk2 expression was enhanced in PARK2 astrocytes and midbrain organoids, confirming the previous finding that Park2 mutations lead to KAR upregulation (Maraschi A, 2014). Neuronal reactivity was also found increased in PARK2 midbrain organoids at electrophysiology assessment, maybe linked to glutamate dysregulation. Two innovative findings emerged from this study. First of all, that TH expression resulted increased in PARK2, supporting previous finding that stated an augmented dopamine turnover and a reduced dopamine re-uptake (Jiang H., 2012). This is an impairment that happens early in the neurodegenerative process and that could consequently lead to an excessive oxidative stress and consequent neurodegeneration. The second original result is that PARK2 is associated to an increased astrocytic reactivity and a possible dysfunction of astrocytic glutamate transporter EAAT2. This finding means that astrocytes play a key role in neurodegeneration although it is not clear whether they contribute to the initiation or propagation of it. Their increased reactivity could be the consequence of a glutamate toxicity or glutamate toxicity could result from reactive astrocytes dysfunction, not able to process the excessive glutamate influx. Further studies are required in order to establish Park2 role in TH expression regulation, in astrocytic reactivity induction and in glutamate toxicity.

Human pluripotent stem cells are quickly emerging as a fundamental tool for in vitro studies. In particular, the advent of “induced pluripotency” opened completely new horizons for in vitro disease modeling and patient-specific disease-on-a-dish therapeutic approach screening. The easy access to cell types of human origin hardly available otherwise, with virtually infinite amounts in a donor-unrestricted manner, unlocked in vitro studies for human tissues such as brain, pancreas and the heart. In the latter case, the need for new models of human cardiac physiology and physiopathology is highlighted by the severe fallouts of heart conditions on worldwide health and economy. The main focus of this thesis are human cardiomyocytes derived through differentiation of pluripotent stem cells, and their application as an in vitro model of the human cardiac tissue. In particular, the stress point of the work is their early and immature phenotype, that often limits their application and frustrates the potential of a human heart model in a Petri dish. After introducing the current scenario of study models for heart diseases and describing the main features of human pluripotent stem cells (hPSCs) and their cardiac derivatives (hPSC-CMs), this thesis will separately focus on the two main aspects of the cardiomyocyte physiology: structural and functional features and metabolic profile. From these perspectives, human cardiomyocytes derived from hPSCs display in vitro an early and immature phenotype, closely resembling cardiomyocytes at early stage of the development, such as fetal cardiomyocytes. Cell ultrastructural organization and functional performance are two strictly related features that find in adult cardiomyocytes perfect synthesis, with a very specialized function performed through a finely orchestrated sequence of events hugely relying on the right spatial distribution of key molecular components. In Chapter 2, biomaterials and microengineered substrates are employed to address the molecular mechanisms triggering cardiac maturation in vitro, in order to provide insight in the process and drive hPSC-CMs towards more adult-like phenotypes, better suiting disease modeling and drug screening. Cardiac metabolism is likewise a characterizing feature of the tissue supporting in a unique fashion the impressive workload of the heart. In Chapter 3, hPSC-CM metabolism is described and a novel microfluidic technology is developed for metabolic maturation screening of cardiac cultures. With this approach, hPSC-CMs are shown to positively respond to an optimized metabolic maturation protocol, similar to the very rapid fetal-to-adult metabolism switch in hCMs after birth in response to changing metabolite availability. Finally, in perspective of the maturation approaches previously described and their feasible application to human cardiac cultures, in Chapter 4 are discussed two human genetic diseases affecting the heart muscle. For both Duchenne’s muscular dystrophy and arrhythmogenic right ventricular cardiomyopathy/dysplasia, cardiac cellular models are set up and proven to display in vitro the molecular hallmarks of the disease, thus providing the biological substrate for further studies on human cardiomyocyte cultures.
Le cellule pluripotenti umane stanno velocemente emergendo come strumenti fondamentali nella ricerca in vitro. In particolare, l’avvento delle cellule pluripotenti indotte ha aperto nuovi orizzonti sullo studio e la modellazione delle malattie umane e lo screening di approcci terapeutici. La possibilità di avere in coltura cellule di origine umana provenienti da tessuti da cui è difficile ottenere campioni bioptici, ha permesso di delineare nuove prospettive di studi in vitro per tessuti come il cervello, il pancreas o il cuore. Specialmente in quest’ultimo caso, il bisogno di nuovi modelli di studio è esaltato dall’impatto che le malattie cardiache hanno sulla sanità e sull’economia mondiali, a cui i modelli di studio tradizionali non riescono a far fronte in modo efficace. L’oggetto di studio di questa tesi di dottorato sono i cardiomiociti umani derivati per differenziamento da cellule staminali pluripotenti e la loro applicazione come modello di studio del tessuto cardiaco. In particolar modo, ci si è focalizzati sul loro fenotipo, ritenuto precoce ed immaturo rispetto al cardiomiocita adulto, che limita enormemente il loro impiego in campo medico e scientifico, impedendo il pieno sviluppo del loro potenziale. Dopo aver introdotto i modelli sperimentali attualmente impiegati nello studio di patologie cardiache, verranno descritte le principali caratteristiche delle cellule pluripotenti umane (hPSC) e dei loro derivati cardiaci (hPSC-CM). Successivamente, l’attenzione verrà focalizzata su due caratteristiche fondamentali che descrivono la fisiologia di un cardiomiocita: l’organizzazione strutturale della cellula legata alla sua funzionalità ed il suo profilo metabolico. In entrambe queste categorie, i hPSC-CM vengono spesso paragonati a cellule cardiache fetali, lontane in termini di sviluppo dal fenotipo del cardiomiocita adulto. L’organizzazione ultrastrutturale di una cellula cardiaca è strettamente correlata con la sua capacità funzionale: nei cardiomiociti adulti si assiste ad una perfetta concertazione spazio-temporale di diverse componenti molecolari la cui azione coordinata permette alle cellule del cuore di svolgere l’attività contrattile. Nel Capitolo 2 di questa tesi, vengono impiegati biomateriali e substrati micro- ingegnerizzati per studiare i meccanismi molecolari che promuovono la maturazione strutturale e funzionale dei cardiomiociti umani in vitro, rendendoli così più vicini fenotipicamente ad una cellula adulta su cui svolgere studi farmacologici e di modellazione di patologie. Il metabolismo cardiaco è una caratteristica altrettanto unica e caratterizzante per un cardiomiocita, essendosi adattato ad sostenere un’attività costante ed energicamente dispendiosa com’è la generazione di forza meccanica. Nel Capitolo 3 viene descritto il metabolismo di un cardiomiocita e viene proposta una nuova piattaforma microfluidica da utilizzare per la validazione di protocolli di maturazione metabolica in vitro. Con il saggio funzionale messo a punto viene inoltre dimostrato come l’induzione di un metabolismo maturo è possibile attraverso la variazione dei substrati energetici presenti nel mezzo di coltura. Infine, in prospettiva dell’applicazione dei protocolli di maturazione descritti nei capitoli precedenti a colture cardiache umane, nel Capitolo 4 viene presentato l’allestimento di due modelli cellulari di patologie genetiche: la distrofia muscolare di Duchenne e la cardiomiopatia aritmogena del ventricolo destro. Vengono presentati e caratterizzati cardiomiociti umani derivati per differenziamento da cellule hiPS di pazienti affetti, mostrando come i cardiomiociti in coltura presentino il fenotipo molecolare aberrante caratterizzante le malattie prese in esame. Si fornisce, così, un modello cellulare cardiaco umano che può trovare impiego nella modellazione in vitro delle due patologie.

Diabetic neuropathy one of important complications of diabetes, is associated with neuropathic pain in about 50% of diabetic subjects. Clinical management of neuropathic pain is complex and so far unsatisfactory. To this aim in rats rendered diabetic by streptozotocin injection we have analyzed the effects of the testosterone metabolites, dihydrotestosterone (DHT) and 5α- androstane-3α,17β-diol (3α-diol), on nociceptive and allodynia thresholds. Moreover, molecular and functional parameters in the spinal cord related with pain modulation, such as the levels of glutamate, the expression and phosphorylation of synaptic proteins, the expression of substance P, neuroinflammatory markers and translocator protein, as well as the number of GFAP immunoreactive astrocytes have been analyzed. Finally, the levels of DHT and 3α-diol have been evaluated in spinal cord of steroid treated and untreated animals. Diabetes resulted in a significant decrease in DHT levels in the spinal cord that was reverted by DHT or 3α-diol treatments. In addition, 3α-diol treatment resulted in a significant increase in 3α-diol in the spinal cord over control values. Both steroids show analgesic properties on diabetic neuropathic pain. Interestingly, they exert their effects affecting different pain parameters and possibly by different mechanisms of action. Indeed, DHT treatment counteract the effect of diabetes on mechanical nociceptive threshold, pre- and post-synaptic components, glutamate release, astrocyte immunoreactivity and expression of interleukin-1β, while 3α-diol treatment was effective on tactile allodynia threshold, glutamate release, astrocyte immunoreactivity and the expression of substance P, toll-like receptor 4, tumor necrosis factor-α, transforming growth factor β-1, interleukin-1β and translocator protein. Moreover a DRG primary cell colture experiment was performed in order to demonstrate the 3α-diol specific effect GABA-A-mediated on substance P expression. Altogether these results suggest that testosterone metabolites are potential agents for the treatment of diabetic neuropathic pain.

Idiopatic Central Hypogonadism (ICH) is a rare pathology with a strong genetic component, in which hypothalamic and pituitary dysfunctions involving development and/or functionality of GnRH neurons, cause a reduced or absent gonads functionality. This disease can occur in association with anosmia or hyposmia, (Kallmann Syndrome, KS) o with a normal sense of smell (normosmic idiopathic hypogonadotropic hypogonadism, nIHH) and it shows an extreme phenotypic variability. Despite the identification of 14 genes implicated in the pathogenesis of the disease, approximatively 70% of ICH cases remains idiopathic. Among the causative genes, a role of particular importance is covered by the the Prokineticin pathway, in particular the Prokineticin-2 (PROK2) and its receptor (PROKR2). In fact, in approximately 10% of cases of ICH is possible to identify a genetic variant in one of these two genes such as pathogenetic event of the disease. The receptor PROKR2 belongs to the family of G-protein coupled receptor (GPCR). Its activation, through the binding with PROK2, determines the activation of protein Gq, Gs and Gi, a consequent production of IP3, cAMP and subsequently the mobilization of intracellular calcium. To date in literature have been described 27 PROKR2 mutations and the functional studies performed on a minority of them have only evaluated the effects on the Gq-IP3 signal transduction pathway. Nevertheless a growing number of works in the field of GPCRs demonstrates the importance of the functional studies of all the possible pathways related to a single receptor in order to interpret the functional consequences of genetic variants identified. In the present work we have developed two main lines of research starting from the wider availability of Italian cohort of ICH patients. In the first part of this work we have carried out studies of genetic screening of a cohort of 217 patients, considering the main causative known genes for that pathology, including PROKR2. Genetic variants identified in PROKR2 were then characterized by a functional point of view to test their potential pathogenic role. This screening allowed the identification of seven PROKR2 missense variants (V158I, L173R,T260M, R268C, V274D, V331M and V334M) of which 3 have not yet been described in the literature; in addition to 2 variants nonsense (15fsX45, 20fsX43). The variants identified have been inserted by site-directed mutagenesis into vectorsSPRT-PROKR2-pcDNA3, characterized by the presence of a Rhodopsin tag at the N-terminus of the receptor. This allows the display of the cellular localization of the mutants by binding with an anti-rhodopsin antibody. The constructs thus generated were transfected into HEK 293 cells and CHO for the following functional studies. The FACS analysis revealed that all variants have a reduced membrane expression (reduction of 11-55%), with the exception of the mutation V334M, which shows an expression slightly exceeding that of the wild-type receptor. Functional assays were then performed with the generation of concentration-effectcurves for both IP1 that for cAMP. The results obtained show how the mutation T260M, R268C, V274D, V331M and V334M cause a strong reduction of the signal mediated by the Gq protein , while the signal of cAMP mediated by the Gs protein is significantly reduced in the mutant L173R andV334M. The V334M and V274D mutations are characterized by a marked inactivation of both pathways. Finally analyzing the homology model of PROKR2 it appears evident that the variant V331M is localized at the level of a highly conserved domain (the motif NPXXY), involved in signal transduction. These are the first experiments that analyze both transduction pathways activated by PROKR2 receptor and showing how the different variants associated with ICH can affect signal transduction pathways in a very variable manner. In particular, some variants causing inability to stimulate the two pathways, suggesting that the integrity of both is necessary for normal development and function of GnRH-secreting neurons. The second part of this thesis, it was instead intended to further clarify the genetic mechanisms (and eventually epigenetic) about the ethiopathogenesis underlying ICH. To conduct these studies we used the techniques of SNPs and CNVs genotyping , on a selected series of familial cases of ICH. For each patient were analyzed 660,000 SNPs and CNVs 100,000, then comparing them with a large database of apparently healthy controls in our possession, in a case / control analysis. The analyzes focused on the identification of SNPs and on the presence of CNVs significantly correlated with ICH and on an family type analysis to detect extended regions of homozygosity in patients (LOH = Loss of Heterozigosity regions). A first macroscopic analysis of the data obtained shows that the number and extent of the deletions in single copy is significantly higher in cases of ICH, compared to controls. The analysis of SNPs and CNVs showed, among the 30 identified loci four genes (CNTNAP2, GPC, RAB39B and PPFIA2) that for expression and molecular function seem to be good candidates for direct sequencing screening in ICH patients. Furthermore we have identified three clusters of microRNA (mir4275, mir507/508/509,mir320D2), suggesting for the first time a potential involvement of these molecules in ICH pathogenesis. Analyzing instead the genes that reside in LOH areas , it is possible to observe an enrichment for certain pathways or protein families, such as: the FGFR pathway; cadherins and cell adhesion molecules (CAM); receptors and ligands involved in the differentiation of the central nervous system (CNS), hypothalamus and pituitary; genes associated with midline defects or with Prader-Willi and Angelmann syndrome; plexins and RAB proteins.

Using biochemical and genetic approaches we have been able to identify the genetic defect underlying mitochondrial complex I deficiency in 3 patients out of the 12 patients in the cohort. The patient cohort investigated in this study is genetically heterogeneous, originating from several different geographical areas: England, France, the Middle East, Israel, India and Pakistan. They presented with different clinical phenotypes: Leigh syndrome, congenital lactic acidosis, hypertrophic cardiomyopathy, encephalopathy, developmental delay, Alpers’ disease. After having excluded the mitochondrial DNA molecule for carrying mutations in muscle (analysis carried out by the diagnostic laboratories’ staff, at the National Hospital for Neurology, Queen Square, London), a biochemical investigation was undertaken in patients’ fibroblasts: the complex I activity defect was confirmed in this tissue as well for all the patients, and Blue Native studies were carried out. Antibodies against several subunits of complex I identified various subassemblies of the ~1MDa holoenzyme in several patients; in some others various degrees of reduction in holo-complex I content were observed. Analysis of the complex I pattern on Blue Native gels led the genetic screening towards a subset of genes, already known to be involved in complex I deficiency. Two families were also run on the Affymetrix 10K SNP chip array and then a homozygosity mapping approach was undertaken on the assumption that the affected individuals inherited two copies of the same ancestral mutated allele from a common ancestor (autozygosity). A subsequent bioinformatics analysis (also involving the implementation of the Maestro and MitoCarta databases) allowed the selection of a subgroup of genes that could possibly bear the genetic defect; this was done taking into account the Blue Native complex I pattern as well. Genetic screening identified a novel 8bp frameshift deletion (c.377_384del; Q126fsX2) in the NDUFS4 gene as cause of the disease in a patient from the first pedigree analyzed by homozygosity mapping. Her cousin was heterozygous for the same defect, but no other mutation has been identified, leaving this complex I deficiency case unsolved. In the second pedigree analyzed by homozygosity mapping approach, a homozygous mutation in a novel complex I assembly factor never previously linked to human disease has been identified. The c.1054C>T; R352W mutation in FOXRED1 segregated with disease in the family and was not found in 268 healthy control alleles. Western blot analysis showed a reduced steady-state level of FOXRED1 in patient fibroblasts and restoration of complex I activity after lentiviral transduction of patient fibroblasts with wild-type FOXRED1 cDNA. Finally, by candidate gene sequencing, two novel compound heterozygous mutations in NDUFAF1 were identified in a third patient: c.631C>T; R211C and c.733G>A; G245R. In summary, this study allowed the identification of mutations in (i) one complex I subunit, NDUFS4, already associated with complex I deficiency; (ii) one novel complex I assembly factor: FOXRED1; (iii) and NDUFAF1, a known complex I assembly factor whose mutations give a similar complex I Blue Native pattern to the one observed in our patient. In conclusion our combined approach proved to be efficient in the identification of the genetic defect in patients affected with complex I deficiency.

Osteoarthritis (OA) is a highly disabling pathology which is worldwide investigated by the scientific community due to its increasing diffusion. The incidence of this age-related disease is increasing with population ageing and worldwide estimates indicate that 9.6% of men and 18% of women with more than 60 years present OA-related symptoms. Osteoarthritis induces the progressive damage of articular cartilage and subchondral bone and can eventually lead to the complete loss of joint functionality. Nowadays, the management of OA includes non-pharmacological, pharmacological, and surgical treatments. Non pharmacological approaches include exercise, weight loss, and physiotherapy and are used in conjunction with pharmacological treatments. The pharmacological management of OA patients is mainly based on the use of anti-inflammatory drugs for pain control. Hence, the pharmacological approach to OA patients is synptomatic, but not resolutive, since it is not able to alter the progression of the disease. These therapeutical options are not suitable for late stage OA patients, whereby the severe pain and the functional limitations caused by advanced OA degeneration are currently resolved by joint replacement. Due to the low self-repair ability of articular cartilage, untreated cartilage lesions can easily degenerate into OA. Different strategies have been developed to treat promptly chondral lesions, comprising cell-based therapies, such as autologous chondrocyte implantation (ACI). These cell-based therapies have been recently proposed also for the treatment of early OA patients in order to overcome or at least delay the need for a more invasive intervention such as joint replacement. However, major issues associated to the clinical use of autologous articular chondrocytes (ACs) are the limited number of cell harvestable from a small cartilage biopsy and the de-differentiation process occurring during the expansion phase required to achieve a clinically relevant number of cells. Furthermore, advanced age and pathological state of joints negatively affect the chondrogenic potential of ACs, representing important factors to be considered when applying chondrocyte-based therapies in particular categories of patients. In view of a possible use of autologous cell-based therapies for OA patients, we analyzed specific features of ACs as cellular yield, cell doubling rates and the dependence between these parameters and patient-related data in a set of 211 OA patients undergoing total joint replacement (Chapter 3). The patient age was not statistically correlated to the cellular yield, but was negatively correlated with the proliferation rate. No significant correlation was observed between the level of cartilage degeneration (ICRS score) and cellular yield and proliferation rates. However, in samples with the highest degree of cartilage degeneration (ICRS score 4) the cellular yield was lower compared to the other three groups (ICRS scores 1-3). In conclusion, we found that age and degenerative state of cartilage affect some basic parameter of articular chondrocytes and should be considered when evaluating the quality of the cell source to be used in clinical applications. To overcome some of the limitations related to the use of ACs, mesenchymal stem cells (MSCs) present in several tissues, such as bone marrow and fat, have been proposed as cell candidate for cartilage treatment thanks to their demonstrated chondrogenic ability. When developing treatments for knee chondral lesions, infrapatellar fat pad and knee subcutaneous adipose tissue, two fat depots easily accessible during knee surgery, can be considered appealing sources for MSCs harvesting. We performed a donor-matched comparison between infrapatellar fat pad MSCs (IFP-MSCs) and knee subcutaneous adipose tissue stem cells (ASCs) obtained from OA patients undergoing total knee replacement, analyzing their immunophenotype and multi-differentiative ability, with a focus on their osteogenic and chondrogenic potential (Chapter 4). We found that these cell populations share common features at the undifferentiated state, such as immunophenotype and clonogenic potential, but display a specific commitment towards the osteogenic and chondrogenic lineage. Indeed, significantly higher levels of osteogenic markers, as calcified matrix deposition and alkaline phosphatase activity, were found in ASCs, highlighting the superior osteogenic commitment of this cell population in comparison with IFP-MSCs. Conversely, IFP-MSCs differentiated towards the chondrogenic lineage by 3D pellet culture showed greater glycosaminoglycans (GAGs) deposition and higher expression of chondrogenic genes as aggrecan (ACAN) and type II collagen (COL2A1) compared to ASCs pellets, revealing a superior chondrogenic potential. This result was supported by lower expression of hyperthrophic and fibrotic markers, as type X (COL10A1) and type I (COL1A1) collagen, in IFP-MSCs pellets compared to ASCs. The observed dissimilarities indicate that populations of adipose-derived MSCs harvested from different anatomical sites have specific features that suggest their preferential use for specific cell-based applications. In the last decade, the co-culture of ACs and MSCs has gained growing interest to investigate the cross-talk between these cell types and to evaluate the possibility of replacing a fraction of ACs with MSCs, in order to reduce the in vitro expansion phase of ACs and subsequently limit their de-differentiation. Controversial results have been reported on the possibility to generate cartilage-like matrix by co-culturing ACs and MSCs, indicating a prominent role for the origin and de-differentiation state of chondrocytes as determinants of the outcomes. We evaluated whether the co-culture of IFP-MSCs and ASCs with a small fraction of ACs was able to lead to cartilage-like matrix generation and to the expression of chondrogenic markers comparable to ACs (Chapter 5). To better resemble a possible clinical application of this strategy we performed autologous co-cultures combining IFP-MSCs and ASCs with expanded and cryo-preserved donor-matched ACs and we used cells derived from OA patients so as not to neglect the impact of the pathological and de-differentiated state of ACs on the outcome of the co-cultures. Chondrogenic genes SRY (sex determining region Y)-box 9 (SOX9), COL2A1, and ACAN were less expressed in co-cultures compared to ACs mono-cultures. No significant differences were observed for GAGs/DNA in mono-cultures, demonstrating the reduced chondrogenic potential of ACs, probably deriving by both their pathological origin and their de-differentiated state. Total GAGs content in co-cultures did not differ significantly from values predicted as the sum of each cell type contribution corrected for the co-culture ratio, as confirmed by histology. Therefore, a small percentage of expanded and cryopreserved ACs did not lead to IFP-MSCs and ASCs chondro-induction. Our results suggest that chondrogenic potential and origin of chondrocytes play a relevant role in the outcome of co-cultures, indicating the need for further investigations to demonstrate their clinical relevance in the treatment of aged osteoarthritic patients. As aforementioned, chondrogenic differentiation of both ACs and MSCs is usually performed in 3D culture models that partially mimic the 3D environment of cartilagineous matrix such as culture in pellets and in 3D scaffolds. Pellet culture is widely used in the field of cartilage tissue engineering to test and optimize differentiation protocols. These experiments normally require to prepare replicates from a relevant number of donors to investigate different culture conditions. Unfortunately, handling a high number of samples in 3D pellet culture is extremely time consuming and is a major issue in experiments with many different conditions. To overcome these limitations, we exploited low-cost rapid prototyping techniques, such as laser ablation and replica molding, to generate multi-well chips in polydimethylsiloxane (PDMS) for the formation and culture of cell aggregates (Chapter 6). Each PDMS chip allowed the simultaneous culture of several pellets leading to a significant reduction in the time required for pellet seeding and medium refresh operations. Proliferation and metabolic activity were comparable between pellets of ACs cultured in PDMS chips and pellets cultured in polypropylene tubes, whereas type II collagen deposition was increased in pellets cultured in the PDMS chips. The same rapid prototyping approach was applied to generate a patterned scaffold. As a proof of concept, we biofabricated a multi-well implantable constructs using clinically approved fibrin glue. We demonstrated that regions with different cell densities can be generated within the construct and that cells can be distributed in a spatially controlled way. These multi-well implantable scaffolds can be seeded with multiple cell types allowing the precise distribution of different cell populations, thus representing a useful tool to investigate in vitro and in vivo cell interactions in a 3D environment. The introduction of engineered tissue into the clinical practice implies the achievement of high quality and safety standards. The use of automated bioreactor-based manufacturing can significantly increase the reproducibility of the results, being minimally affected by operator variability. Furthermore dynamic culture systems, such as perfusion bioreactors, represent a smart approach to overcome the limitations encountered in the static culture of 3D constructs having clinically relevant dimensions. Indeed, perfusion bioreactors have been shown to increase the homogeneity of cell distribution within the scaffold compared to static cell seeding. Perfusion culture provides a continuous and homogeneous influx of fresh medium throughout the construct, improving the quality and homogeneity of the extracellular matrix. The use of optimized protocols for the expansion and re-differentiation of ACs employing clinically approved growth factors is another key step to achieve a successful outcome and to grant the clinical translability of the results. Thus, in collaboration with the group supervised by Prof. Frédéric Mallein-Gerin (Institut de Biologie et Chimie des Proteines, Lyon, France) we tested the combination of a specific cocktail of clinically approved growth factors with a bi-directional perfusion bioreactor (OPB, Oscillating Perfusion Bioreactor) with the aim of improving cartilage matrix production (Chapter 7). We established a perfusion program including phases of high and low perfusion speeds to alternate sequences of cell stimulation and matrix deposition and we compared collagen sponges seeded and cultured in dynamic conditions with sponges seeded and cultured in standard static conditions. We found that perfusion improved cartilage matrix deposition within the sponges, in comparison with static conditions. More precisely, in the sponges cultured in the bioreactor a cartilaginous matrix rich in type II and type IX collagen and GAGs, with no signs of hypertrophy, was produced. Furthermore, a lower amount of type I collagen was produced in the sponges cultured in dynamic conditions, indicating that perfusion limits the risk of fibrocartilage formation. In conclusions, the dynamic culture by means of a perfusion bioreactor combined with the use of a cocktail of clinically approved growth factors proved to be effective for the generation of engineered cartilagineous grafts, improving the homogeneity and quality of the extracellular matrix generated by articular chondrocytes seeded within collagen sponges. Aged patients with late stage OA involving an extensive erosion of articular cartilage and exposure of the underlying subchondral bone cannot be treated with cell-based therapies or standard surgical approaches aiming at cartilage restoration. In these patients, joint replacement is currently the only clinical option to restore the articular function. The insufficient implant stability is an important determinant in the failure of cementless prostheses, leading to an increase in the risk of implant mobilization and in the number of revision procedures, with major drawbacks for patients and for National Health Systems. With the aim of increasing implant osseointegration, porous metallic materials have been developed and applied to cementless implant technology to maximize bone ingrowth and bone-implant contact. More recently, the enrichment of porous titanium with growth factors has been proposed as a novel strategy to further improve implant osseointegration. We combined a macroporous titanium (Trabecular Titanium™, TT), currently used in the clinical practice, with a biocompatible hydrogel (amidated carboxymethylcellulose, CMCA) able to encapsulate osteoinductive factors and osteoprogenitor cells (Chapter 8). In particular, we chose strontium as osteoinductive factor and bone-marrow derived MSCs (BMSCs) as osteogenic progenitor cells for implant enrichment. We tested different concentrations of SrCl2 to select the optimal concentration to induce BMSCs differentiation. We found that SrCl2 at 5 µg/ml significantly increased calcified matrix deposition, type I collagen (COL1A1) expression and alkaline phosphatase activity, being the most effective concentration among the ones tested. The enrichment of TT with CMCA (TT+CMCA) significantly increased cell retention within the implant, representing an important improvement in view of a future clinical application of the bioactive implant. Furthermore, we found that BMSCs cultured in TT+CMCA in the presence of SrCl2 underwent a more efficient osteogenic differentiation, as demonstrated by higher alkaline phosphatase activity and calcium levels. Based on these in vitro results, we performed an in vivo study to evaluate the performance of the bioactive implant generated combining the macroporous titanium with strontium-enriched CMCA and BMSCs (Chapter 9). To mimic implant-bone interaction, an ectopic model was developed grafting TT implants into decellularized bone seeded with human BMSCs. TT was loaded or not with strontium-enriched CMCA and/or BMSCs and constructs were implanted subcutaneously in athymic mice. Osteodeposition at the bone-implant interface was investigated with fluorescence imaging, micro-CT, scanning electron microscopy (SEM), histology and biomechanical testing at different time points. Micro-CT analysis demonstrated the homogeneity of the engineered bone in all groups, supporting the reproducibility of the ectopic model. Fluorescence imaging, histology, SEM and pull-out mechanical testing showed superior tissue ingrowth in TT implants loaded with both strontium-enriched CMCA and BMSCs. In our model, the synergic action of the bioactive hydrogel and BMSCs increased both bone deposition and TT integration, indicating that the generation of bioactive implants able to promote bone deposition is a promising strategy for the improvement of implant osseointegration. Joint inflammation is an important trait of osteoarthritis, indeed synovitis is observed in various degrees in OA patients, with OA synovium showing a mixed inflammatory infiltrate mainly consisting of macrophages. The central role of macrophages in OA evolution supports the interest in investigating their response to signals typical of OA joints, such as the ones contained in the synovial fluid (SF) of OA patients. In collaboration with the Connective Tissue Cells and Repair group supervised by Prof. Gerjo J.V.M. van Osch (Erasmus MC, Rotterdam, The Netherlands), we investigated the effect of SF from OA patients on the transcriptional expression of anti-inflammatory and pro-inflammatory mediators in monocyte-derived macrophages (Chapter 10). SF from donors without any joint pathology was used as control, and the effect of the different types of SF was tested on non-activated macrophages (M0) and on macrophages activated by IFNγ and TNFα (M1 activation) or IL4 (M2 activation). We found that IL10 and IL1ra were modulated by the treatment with OA SF in M0 and M2 conditions. The effect on IL10 was lost in M1 conditions probably due to the high up-regulation induced by the treatment with IFNγ and TNFα. Our results support the idea that arthritic SF is for macrophages a less pro-inflammatory environment compared to control SF. This may mimic the macrophage situation in the joint, where feedback mechanisms are induced to counteract the ongoing pro-inflammatory processes. Altogether our findings highlight the complexity and multiplicity of factors that should be considered for the development of successful cell based treatments for early and late OA patients. We found that basic features of articular chondrocytes were affected by donors age and cartilage degeneration. We also found that expanded OA ACs did not exert a significant chondroinductive effect on co-cultured MSCs, suggesting that the evaluation of the clinical relevance of this approach in OA patients should strongly consider the impact of the de-differentiation state and pathological origin of ACs. Further investigations in this field are needed to verify whether it is possible to replicate the results obtained using non-expanded healthy ACs to co-cultures using expanded ACs from aged patients affected by osteoarthritis. These investigations may require the optimization of clinically approved protocols for expansion and re-differentiation of OA chondrocytes as well as the implementation of three-dimensional and dynamic culture systems. We demonstrated that low-cost rapid prototyping techniques are a useful tool to develop high-throughput systems for the culture of 3D cell aggregates and to generate patterned scaffolds that allow the controlled spatial disposition of different cell types. We also showed that the use of perfusion bioreactors for the dynamic culture of 3D constructs can improve the quality of the engineered tissue, granting a more homogeneous and efficient distribution of nutrients and cells throughout the construct. In order to overcome the limitations related to the use of ACs, MSCs can be considered as promising alternative cell candidates. Here, the identification of specific subsets of MSCs displaying a peculiar commitment should drive the selection of the most suitable cell source. Indeed, as we demonstrated for IFP-MSCs and ASCs, despite common features, specific populations of MSCs harvested from different anatomical sites have intrinsic features that make them more or less suitable for a specific application. Cell-based therapies aiming at cartilage restoration cannot be currently applied to late stage OA patients. However, in this category of patients cell-based approaches can be developed to face issues that are critical in implant technology, such as poor osseointegration. As we proposed here, osteoprogenitor cells, osteoinductive factors, and macroporous metals can be combined to generate a bioactive implant with improved osseointegration ability. This approach could be used to generate “off-the-shelf” implants pre-loaded with osteoinductive factors that can be seeded with autologous BMSCs with a fully intra-operative approach. Finally, when developing cell-based approaches for OA patients, the inflammatory state of the OA joint cannot be neglected. We found that macrophage transcriptional expression was affected by OA synovial fluid. This suggests that OA environments may also induce alterations in the re-implanted cells and highlights the need for better in vitro models to evaluate the response of cells and engineered tissues to a diseased environment. In conclusion, to develop novel cell-based approaches for the treatment of OA basic and translational studies should take into account multiple factors that may affect the clinical outcome, such as the impaired phenotype of OA chondrocytes, the peculiar commitment of specific populations of MSCs, and the impact of inflammatory environment of OA joints.

Background. In the last years tissue engineering for cardiac pathologies has been broadly developed with the aim to restore or improve the diseased or damaged heart. Novel cardiac tissue engineering approaches combine the use of biocompatible scaffolds with stem cells to conjugate material science, surgery and cell therapy techniques. So far, different kinds of stem cells have been described and their potential for cardiac regeneration broadly investigated. We have previously described that it is possible to derive lines of broadly multipotent cells from the amniotic fluid (Amniotic Fluid Stem cells; AFS cells). The aim of this study was to characterize more in detail the AFS cells cardiomyogenic potential both in vitro and in vivo. Methods. Neonatal rat cardiomyocyte (rCM) cells were obtained by enzymatic digestion of 2-3-days old rat hearts. GFP-positive rat AFS (gfp+rAFS) cells were obtained from amniotic fluid samples from GFP-positive transgenic pregnant rats. Human AFS (hAFS) cells were obtained from healthy amniotic fluid back up samples from prenatal diagnosis, following informed consent. AFS cells were isolated by immunosorting for the stem marker c-kit. Before applying a tissue engineering approach, using biocompatible scaffolds, to the AFS and rCM cells coculture, the AFS cells “cardiomyocyte-like” phenotype, acquired in cocolture, had been functionally evaluated by patch-clamp analysis. In this work two different kinds of bidimensional micropatterned scaffolds were used: hydrogel films and PDMS (silicon) membranes. The scaffolds were obtained by microcontact printing technique and using a mold scratched with the desidered micropattern and their viability was tested using, at first, the rat neonatal primary culture. AFS and rCM cells were seeded together on the micropatterned PDMS membranes and analyzed for the expression of troponin T by immunostaining after 6 and 10 days of culture. For the in vivo study, immunodeficient nude male rats underwent a cryoinjury on the heart left ventricle with a 3D collagen scaffold implantation and 5x10e6 hAFS cells/animal local or systemic injection after 15 days. hAFS cells were previously labelled with the red intracellular fluorescent dye CMTMR. Animals were sacrificed at 24 hours, 15 and 30 days after cells injection and hearts stained for cardiac and inflammatory markers. For the acute myocardial infarct model, male Wistar rats underwent an ischemic injury by left anterior descendent coronary artery ligation for 30 minutes and then they were reperfused injecting via the external jugular vein 10e7 or 10e6 gfp+rAFS and 10e7 or 5x10e6 hAFS cells/animal for 2 hours; rats were sacrificed afterwards and hearts analyzed for infarct size measurement by Evans blue staining, by 2,3,5-triphenolltetrazolium chloride (TTC) staining and planimetry with the software Image J. Heart, lungs, spleen and liver were analyzed as well by immunostaining for evaluating hAFS cells content. hAFS cells were also analyzed for the presence of a subpopulation of cardiac progenitors, by RT-PCR analysis, for the expression of early cardiac commitment genes as Isl1 and Kdr. The cells were then studied by ELISA essay to speculate if they can secrete in the culture medium the protein thymosin beta 4, paracrine and cardioprotector factor. Results and Conclusions. Regarding the in vitro results, AFS cells were demonstrated to express a “pace maker cell-like” action potential, when cocultured with rat neonatal cardiomyocyte cells. Moreover, when cultured on the bidimensional scaffold, AFS cells showed to follow the longitudinal orientation of the microstruttured membrane, expressing beating activity and the cardiac protein troponin T. Our in vivo data revealed that hAFS cells, injected into the cryoinjured rat heart, survived in the host up to 30 days, moved from the injection site to the lesioned area in the heart and gave rise to new chimeric capillaries in the patch and cryoinjury area. In the acute myocardial infarct model the results obtained suggested that hAFS cells could exert a paracrine effect in vivo, decreasing the infarct size (measured as the ratio between the infarct area and the ischemic area at risk of necrosis) from a 53,9 ± 2,3% (obtained in control animals receiving PBS injection) to 40,0 ± 3,0% of the ischemic area. Furthermore, hAFS cells were also demonstrated to have a subpopulation of cardiac progenitors, positive for the expression of the early cardiac commitment genes Isl1 and Kdr and to to secrete in the culture medium thymosin beta 4, a paracrine factor previously shown to act as cardioprotector and angiogenic agent. In conclusions, our results are very encouraging and challenging, suggesting that AFS cells can show cardiomyogenic potential and cardioprotective therapeutic application in cell based therapy tissue engineering.

L'inquinamento è uno dei problemi maggiri della nostra società. In particolar modo, l'inquinamneto relativo all'acqua sta diventando sempre più consistente considerando la cresita esponenziale del fabbisogno mondiale. Si stima che nel 2050 la domanda di acqua cresca fino al 50%. In questo scenario è necessario trovare una soluzione a tale problema. in questa tesi viene proposto un metodo innovativo per la fabbricazione di materiali polimerici additivati con nanoparticelle che sono in grado di degradadare inquinanti organici in acqua atterevro il processo della fotocatalisi. Questo metodo permette di degradare direttamente la sostanza inquinante in acqua, ovviando a diversi problemi relativi ai costi e al recupero post-trattamento del materiale utilizzato per la decontaminazione.
Water plays a vital role in sustain life on earth. However, the quality of the water resources is seriously affected by the pollution caused by industrial, municipal and agricultural activities. To remediate the toxic organic and inorganic contaminants in water, several physical, chemical and biological water treatment methods are employed. However, current water treatment technologies present limited performance, materials of high cost, and need highly energy consuming infrastructure. In this scenario, the utilization of advanced materials and innovative methods could be helpful for reducing the cost and enhancing the performance of the wastewater treatment. One of most promising method is the Advanced Oxidation Processes (AOPs). The AOPs use semiconductor materials that are activated by the UV or Visible light irradiation that are able to mineralize organic water pollutants. Currently, commercial available photocatalysts are mostly used in slurry or powder form, but this requires post-treatment processes in order to avoid secondary pollution, causing the increase of the overall treatment cost. To face up such limitation and to resolve problems related to the post-treatment process, the immobilization of the photocatalysts on solid compact matrices is a feasible solution. In particular, the use of polymeric matrices as host solid materials may result in a final system that combines the photocatalytic properties of the semiconductor component with the mechanical properties, usability and cost-effectiveness of the polymers.

Resistance to chemotherapeutic agents is well known in patients with hepatocarcinoma. Inhibition of the mitochondrial permeability transition pore (PTP) is a crucial step in tumor cell resistance to apoptosis induced by anticancer drugs. Since SERPINB3 is overexpressed in hepatocellular carcinoma and has an anti-apoptotic activity, aim of the study was to assess the role of this serpin on PTP modulation during treatment with chemotherapeutic agents. HepG2 cells stably transfected with SERPINB3 where assayed for cell death induced by Cisplatin, Doxorubicin, Etoposide and 5-Fluorine Uracil. Reactive Oxygen Species (ROS) were detected with dichlorofluorescein. Threshold of PTP opening was evaluated by CRC assay and Complex I activity was determined by spectrophotometric assay. After cell death induction by Cisplatin and Doxorubicin, HepG2 cells expressing SERPINB3 showed a significant increase in viability compared to controls, while there was no difference in cell death after Etoposide and 5-FU treatments. The addition of the antioxidant N-acetyl cysteine abrogated cell death induction by Cisplatin and Doxorubicin, suggesting that SERPINB3 protects from death through an antioxidant activity. Since Cisplatin and Doxorubicin induce mitochondrial oxidative stress and favor PTP opening, cells were treated with the PTP inducer EM20-25 that acts at mitochondrial level. In presence of SERPINB3 the effect of EM20-25 resulted in PTP opening inhibition and decreased ROS formation. Subcellular localization analysis revealed that a fraction of SERPINB3 was located in mitochondria and that it increased after death-promoting treatments. Mitochondrial SERPINB3 was found associated to respiratory chain Complex I by immunoprecipitation experiments. In vitro analysis confirmed the inhibition by SERPINB3 of Complex I activity, known as one of the main sources of mitochondrial ROS. In conclusion, SERPINB3 acts at mitochondrial level protecting cells from chemotherapeutic-induced oxidative stress and consequent cell death. This antioxidant function could represent a relevant advantage for a transformed cell to balance its ROS equilibrium and for SB3-expressing cells exposed to anticancer treatments.
L'epatocarcinoma (HCC) è la quinta forma di cancro più diffusa nel mondo ed è caratterizzato da un'alta proliferazione cellulare e da un'alta resistenza ai trattamenti chemioterapici. Negli ultimi anni l'associazione tra la ov-serpina SERPINB3 (precedentemente conosciuta anche come SCCA1) e la progressione della patologia epatica e lo sviluppo di HCC si è fatta sempre più stretta, portando ad ipotizzare che questa proteina possa giocare un ruolo importante nella biologia del tumore epatico. SERPINB3 è in grado di inibire la morte cellulare in sistemi cellulari di tipo epiteliale, ma non si conosce ancora il suo esatto meccanismo d'azione molecolare né la sua funzione nel fegato. Lo scopo dello studio è stato verificare il ruolo di SERPINB3 nella morte cellulare indotta da trattamenti chemioterapici in un modelli epatici sperimentali e di indagarne l'azione a livello molecolare. Esprimendo SERPINB3 in una linea di epatoma (HepG2), si è messo in evidenza come la presenza della proteina riduce la morte cellulare dopo trattamento con Cisplatino e Doxorubicina, ma non dopo trattamento con Etoposide o 5-FluoroUracile. Esperimenti successivi hanno documentato come la resistenza alla morte sia associata ad un diminuito stress ossidativo, definito come riduzione dei livelli di ROS, nelle cellule che esprimono SERPINB3. E' stato dimostrato che la diminuzione dei livelli di ROS si associa alla presenza di SERPINB3 a livello mitocondriale. Utilizzando l'EM20-25, un composto che fa parte della famiglia dei chemioterapici BH3-mimetici, è stato confermato che la protezione viene conferita a livello mitocondriale in quanto viene diminuita l'attività del Complesso I della catena respiratoria con conseguente diminuzione dei livelli di ROS. Questo meccanismo molecolare è stato confermato mediante la documentazione dell'inibizione del Poro di Transizione di Permeabilità mitocondriale da parte di SERPINB3, che è considerato un regolatore chiave dell'innesco della morte cellulare. Dato che SERPINB3 non è presente nel fegato sano, ma la sua espressione progressivamente aumenta in corso di infiammazione cronica e di cirrosi, la sua azione antiossidante, inizialmente protettiva, potrebbe contribuire alla sopravvivenza di cellule trasformate e allo sviluppo di tumore. Nell'ambito dell'epatocarcinoma SERPINB3 potrebbe contribuire alla resistenza ai trattamenti chemioterapici, nota caratteristica di questa forma tumorale epatica.

Background and Aim. Iron is an essential micronutrient, which is involved as a cofactor in fundamental biochemical activities, and it is necessary for proper brain development in the fetal and early neonatal period. However, cellular iron overload produces toxic build-up in many organs, including brain, and, under aerobic conditions, catalyses the propagation of reactive oxygen species and the generation of highly reactive radicals through Fenton Chemistry. Association between metabolic and reproductive impairment has been proved in patients affected by dysmetabolic iron overload syndrome (DIOS). In particular, iron is the most important factor afflicting the hypothalamic-pituitary axis in a dose-dependent fashion leading to hypogonadotropic hypogonadism (HH). Our previous studies in a mouse model of DIOS showed the association between dietary iron overload, visceral adipose tissue insulin resistance and hypertriglyceridemia. Aim of this thesis was to assess whether and how iron overload may affect (a) the reproductive axis (mainly at the hypothalamic-pituitary levels) in a mouse model of DIOS; (b) the migratory feature and GnRH secretory pattern in GN-11 and GT1-7 cells, in vitro models of immature/migratory and mature/GnRH-secreting neurons, respectively. Results. In male mice, dietary-iron overload (IED) led to: a) an increment in testis iron content, b) a reduction in testicular weight and length, c) no changes in hypothalamic iron content c) no changes in mRNA levels of iron-responsive genes, transferrin receptor (TfR) and ferritin H (FtH), in testes and hypothalamus d) an up-regulation of hypothalamic GnRH mRNA levels, e) no changes in hypothalamic Kiss1 and GPR54 gene expression, e) a reduction in pituitary LHβ gene expression. Moreover, the hypothalamic increment of TNFα gene expression along with the phosphorylation/activation of AMPK protein suggested the presence of an inflammatory condition. Increased hypothalamic CHOP mRNA levels also confirmed the endoplasmic reticulum stress feature. IED mice gained less weight than controls showing a reduction in VAT mass and in serum leptin levels, whereas hypothalamic NPY mRNA levels were increased and POMC gene expression was reduced. Western blot analysis showed that the pAkt/Akt ratio was up-regulated in the hypothalamus of IED mice, whereas phosphorylation of ERK1/2 (pERK) protein resulted unchanged in both groups. As far as GN-11 and GT1-7 cells are concerned, a 24-hour treatment with 200 µM Ferric Ammonium Citrate (FAC, source of ferric iron) induced an increment in the intracellular specific iron content of both cell-based models without affecting the cell viability and morphology. Gene expression analysis showed that both cell lines express TfR and FtH, whose mRNA levels were modulated by iron overload. Exposure of GN-11 cells to FAC resulted in the dose (200–1000 µM FAC for 24 hours)- and time (24-72 hours with 200 µM FAC)-dependent inhibition of FBS-induced chemomigration, as assessed by Boyden chamber assay. Pre-treatment with 200 µM deferoxamine (DFO, a specific iron chelator) reverted the above reported iron-driven effect on cell migration. Time-course experiments showed that 200 μM FAC was associated with increased pERK1/2 and pAkt protein levels and with decreased pAMPK ones. Chemomigration assays carried out with the specific inhibitors of ERK1/2, Akt and AMPK highlighted that only Akt pathway seems involved in FAC-mediated inhibition of GN-11 cell migration. In GN-11 cells, iron treatment increased IL-6 gene expression in a dose-dependent mode, whereas NF-kB nuclear translocation and activation was not affected. Up-regulated SOD2 mRNA levels confirmed a condition of activated oxidative stress. Conclusions. The present data show that dietary-iron overload impairs the reproductive axis, probably leading to HH, but further experiments are needed to understand the anatomic site mainly involved in iron-driven damage. Iron treatment negatively affects the migration of GN-11 neuronal cells by the activation of Akt signaling pathway. Hence, iron overload may impair the migration of GnRH neurons from the olfactory placode into forebrain and hypothalamus, where these neurons promote the reproductive competence.

EARLY BIOLOGICAL EFFECTS OF AIR POLLUTION ON CHILDREN: THE RESPIRA STUDY AND THE MAPEC_LIFE PROJECT Elisabetta Ceretti Abstract of the PhD Thesis Background Air pollution is a global problem: airborne or deposited pollutants can be found worldwide, from highly polluted to remote areas. Epidemiological studies attribute the most severe effects from air pollution to particulate matter, which has been associated with cardiovascular diseases, lung cancer and other chronic diseases. In 2013, the International Agency for Research on Cancer (IARC) classified air pollution and particulate matter as carcinogenic to human. Among the whole population, children are at higher risk of suffering the health consequences of airborne chemicals, for various reason. First, children have higher level of physical activity, spend more time outside and have a higher air intake than adults. Second, children are more vulnerable to the adverse effects of air pollution due to their small body size, fast growth rate and relatively immature organs (lungs, in particular), body function, immune system and cell repair mechanisms. Lastly, some data suggest that genetic damage, caused by environmental pollutants, viruses or lifestyle factors, occurring early in life can increase the risk of carcinogenesis in adulthood. Various studies have analyzed the genotoxic effects of urban air pollution exposure in the general population and in highly exposed subjects. In particular, a significant association was found between high levels of urban pollution (PM10 and ozone) and DNA damage detected by the comet assay in human blood lymphocytes and leukocytes and nasal mucosa cells. As regard children, very few data are available on biomarkers of early effect of air pollution. Methods In this research work, some results of two molecular epidemiology cross-sectional projects are presented. Both of them had the objective of evaluating the associations between air pollution and early biological effects in children. The first is the RESPIRA study (Italian acronym for “Rischio ESPosizione Inquinamento aRia Atmosferica”), a small pilot study performed on pre-school children living and attending pre-school in Brescia, a highly polluted town in Northern Italy. The children were recruited in 6 pre-schools located in different areas of the town and their buccal cells were collected to evaluate two biomarkers of early effects: primary DNA damage, detected by comet assay in salivary leukocytes, and micronucleus frequency, investigated in epithelial buccal cells. Child exposure to air pollution was assessed analyzing PM0.5 samples collected near each school in the same days of biological sampling, and retrieving air quality data from the Regional Agency for Environmental Protection. Furthermore, information about some confounding factors was collected by means of a questionnaire filled in by children’s parents. The second study is the MAPEC_LIFE project (Monitoring Air Pollution Effects on Children for supporting public health policy), funded by EU Life+ Programme (LIFE12 ENV/IT/000614) which, in addition to the evaluation of the associations between air pollution and early biological effects in children, aims to propose a model for estimating the global risk of early biological effects due to air pollutants and other factors in children. The MAPEC_LIFE project was carried out on 6-8-year-old children living in five Italian towns in two different seasons. Two biomarkers of early biological effects, primary DNA damage detected with the comet assay and frequency of micronuclei, were investigated in buccal cells of children. Details of children diseases, socio-economic status, exposures to other pollutants and life-style were collected using a questionnaire administered to children’s parents. Child exposure to urban air pollution was assessed by analysing PM0.5 samples collected in the school areas for PAHs and nitro-PAHs concentration, lung toxicity and in vitro genotoxicity on bacterial and human cells. Data on the chemical features of the urban air during the study period were obtained from the Regional Agency for Environmental Protection. The project created also the opportunity to approach the issue of air pollution with the children, trying to raise their awareness on air quality, its health effects and some healthy behaviors by means of an educational intervention in the schools. Results The RESPIRA study involved six pre-schools in Brescia, in two consecutive winter seasons. During the sampling months, PM10, PM2.5 and NO2 were very often over the EU limit values for daily means. Organic extracts of PM0.5 collected near schools induced genotoxic effects in bacterial and human cells in in vitro tests. Regarding DNA damage in children cells, mean micronucleus frequency in epithelial buccal cells of children was 0.29 ± 0.13%, higher than usually found among children living in areas with low or medium-high levels of air pollution, and significantly associated with the concentration of PM10, PM2.5 and NO2. On the other hand, the preliminary results of the comet assay showed some differences between primary DNA damage detected in children attending schools in the different areas of the town. This difference was not detected with the micronucleus test. However, the level of damage resulted from the comet assay on salivary leukocytes of children was not comparable with other literature data, due to the lack of similar studies. The MAPEC_LIFE project involved 26 primary schools in the five Italian towns. Environmental and biological samplings were repeated in the same schools and on the same children for two different seasons, winter and late spring. A total of 1125 children were recruited and sampled for two times. The results of the various in vitro and in vivo tests are still ongoing. Some preliminary results about the samples of the first season in Brescia were presented here. PM10, PM2,5 and NO2 levels remained high for all the winter period, even if they were lower than those registered in the RESPIRA seasons. The organic extracts of PM0.5 collected near each school induced point mutation in Salmonella typhimurium, particularly in the YG1021 strain, but the number of net revertants per cubic meter seem to be slightly lower than other data found in literature. As regard genotoxicity tests on buccal cells of the 283 children recruited in Brescia, no data were available so far for the comet assay, which encountered some reading difficulties. On the other hand, micronucleus frequency detected in MAPEC_LIFE children (0.06 ± 0.08%) was very lower than micronucleus levels found in the RESPIRA study. Statistical analysis of the results is still ongoing, but at the end they will show if the differences in micronucleus frequency between the two studies are due to the level of exposure to air pollution experienced by the children or to other factors. Conclusions The associations between levels of air pollutants, air mutagenicity and biomarkers of early effects will be investigated. A tentative model to calculate the global absolute risk of having early biological effects for air pollution and other variables together will be proposed and may be useful to support policy-making and community interventions to protect children from possible health effects of air pollutants.

(1) Background: Neuroendocrine neoplasms (NENs) are a group of tumors that arise from neuroendocrine cells throughout the body, with the lungs and gastrointestinal tract being the most common sites of origin. In patients with NENs and distant metastases, surgery is generally not curative. Although well-differentiated and low-grade NENs, classified as neuroendocrine tumors (NETs), are usually less aggressive than poorly-differentiated NENs, they can develop distant metastases in about 15% of cases. These patients require chronic medical management. However, the clinical efficacy of these treatments is limited by the low objective response rate, due to the occurrence of tumor resistance and the high biological heterogeneity of these neoplasms. (2) Research problem: We addressed this study on two rare NETs: lung neuroendocrine tumors (LNETs) and medullary thyroid carcinoma (MTC). LNETs represent about 2% of lung tumors, while MTCs are rare thyroid tumors caused by mutations in the RET proto-oncogene. Both NETs are well-differentiated neoplasms and are known to be highly vascularized. Therefore, they represent a potential target for tyrosine kinase inhibitors (TKIs) selective for receptors involved in angiogenesis. The aim of this project was to evaluate the antitumor activity of several new TKIs both in vitro, using LNETs (NCI-H727, UMC-11 and NCI-H835) and MTC (TT and MZ-CRC-1) cell lines, and in vivo, adopting a novel zebrafish xenograft model to study angiogenesis. In LNETs we tested: sulfatinib, a small molecule that inhibits the Vascular Endothelial Growth Factor Receptor (VEGFR) 1, 2, and 3, and the Fibroblast Growth Factor Receptor type 1 (FGFR1); cabozantinib, a multi-target inhibitor selective for VEGFR2, c-Met, Kit, Axl and Flt3; and axitinib, a multi-target TKI of VEGFR1, 2, 3 and Platelet-Derived Growth Factor Receptor-beta (PDGFRβ). In MTC we tested: sulfatinib; SPP86, a RET-specific inhibitor; and SU5402, an inhibitor of the FGFR1 and VEGFR2. (3) Methodology: In LNETs and MTC cells the effects of selected TKIs have been evaluated in vitro through: MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assays, for assessing cell viability; flow-cytometer analysis, for the evaluation of cell cycle and apoptosis; and wound-healing assay, to study cell migration. In vivo we took advantage of the transgenic zebrafish line of Tg(fli1a:EGFP)y1. Through the xenotransplantation of NET cells in the subperidermal space near the subintestinal vein, we assessed the effects of TKIs on tumor-induced angiogenesis and cancer dissemination. (4) Key Results: In LNET cell lines we observed a dose-dependent decrease in cell viability after incubation with all TKIs. This effect seems to be related to the perturbation of the cell cycle and induction in apoptosis. In NCI-H727 wound healing assay showed a significant reduction in cell migration only after incubation with cabozantinib. In the zebrafish model, we found a significant reduction of the tumor-induced angiogenesis in implanted LNET cell lines after treatment with all TKIs. Cabozantinib and axitinib were more potent than sulfatinib in inhibition of angiogenesis, while cabozantinib was the most efficient in reducing cell migration from the transplantation site to the tail. In MTC cell lines, sulfatinib, SU5402 and SPP86 showed a decrease in cell viability, confirmed by the significant reduction in S phase cell population. Moreover, sulfatinib and SPP86 showed for both cell lines a significant induction of apoptosis. Sulfatinib and SPP86 inhibited the migration of TT and MZCRC-1 cells, evaluated through the wound healing assay, while SU5402 was able to inhibit migration only in TT cells. In vivo we observed a significant reduction of TT cells-induced angiogenesis in zebrafish embryos after treatment with sulfatinib and SPP86. (5) Conclusions: Despite sulfatinib resulted the most potent compound in terms of inhibition of LNET cell proliferation, cabozantinib showed in vivo the most effective impact in reducing tumor-induced angiogenesis. Cabozantinib was the only TKI able to inhibit in vivo the dissemination of implanted LNET cells. According to these data, cabozantinib could represent a potential candidate in the therapy of patients with highly vascularized LNET. In MTC cell lines, SPP86 and sulfatinib displayed a similar antitumor activity both in vitro and in vivo, suggesting a good efficacy of specific RET inhibitors (SPP86) with potentially less adverse effects than multitarget TKIs (sulfatinib). In addition, this study showed that the zebrafish model for NETs represents an innovative tool for drug screening with several advantages compared with rodent models: rapidity of procedure, animal immune suppression is not required, lower number of tumor cells for implant and the optical transparency provides a real-time monitoring of cell-stromal interactions and cancer progression in living animals.

My PhD Thesis work, developed in Veneto Nanotech Laboratories (Nanofab in Marghera, LaNN in Padova and ECSIN in Rovigo), was aimed at the exploitation of the Surface Plasmon Resonance (SPR) phenomenon for the set-up of biosensing platforms for clinical and environmental applications. In particular, two types of SPR-based platforms were set-up and optimised: the first one was an oligonucleotide-based platform for the detection of Cystic Fibrosis (CF) causing mutations while the second one was an antibody-based platform for the detection of Legionella pneumophila whole cells. Both sensors are based on the same detection strategy, exploiting the advantages of using a highly sensitive Grating Coupled - Surface Plasmon Resonance (GC-SPR) enhanced spectroscopy method, designed using a conical illumination configuration for label-free molecular detection. Concerning DNA platform for Cystic Fibrosis, a strategy for the detection of some of the most frequent mutations responsible for CF among the Italian population is investigated. For the detection of the CF mutations, gold sinusoidal gratings are used as sensing surfaces, and the specific biodetection is achieved through the usage of allele specific oligonucleotide (ASO) DNA hairpin probes, designed for single nucleotide discrimination. Substrates were used to test unlabeled PCR amplified homozygous wild type (wt) and heterozygous samples (wt/mut) - deriving from clinical samples - for the screened mutations. Hybridisation conditions were optimised to obtain the maximum discrimination ratio (DR) between the homozygous wild type and the heterozygous samples. SPR signals obtained from hybridising wild type and heterozygous samples showed DRs able to identify univocally the correct genotypes, as confirmed by fluorescence microarray experiments run in parallel. Furthermore, SPR genotyping was not impaired in samples containing unrelated DNA, allowing the platform to be used for the parallel discrimination of several alleles also scalable for a high throughput screening setting. Concerning antibody platform for Legionella pneumophila bacteria detection, a strategy for the exploitation of the SPR phenomenon to develop a fully automated platform for fast optical detection of Legionella pneumophila pathogens was investigated. The legal limit of L. pneumophila in a high-risk hospital environment in Italy is 102 CFU/L, and the gold standard for its identification is a time consuming microbiological culture method, that requires up to 7 days. Starting from these considerations a sensitive GC-SPR system was applied to the detection of L. pneumophila to test the detection limit of the developed sensing device in term of detectable bacterium CFU. The detection was accurately set up and precisely optimised firstly through the usage of flat gold functionalised slides to be then translated to sinusoidal gold gratings for label-free GC-SPR detection using ellipsometer, in order to ensure a reproducible and precise identification of bacteria. Through azimuthally-controlled GC-SPR, 10 CFU were detected, while in the case of fluorescence analysis results, a negative readout is obtained if incubating less than 104 CFU. Successful results were obtained when incubating environmental derived samples. This detection platform could be implemented as a prototype in which water and air samples will be sequentially concentrated, injected into a microfluidic system, and delivered to the SPR sensor for analysis. The peculiar Grating Coupled - Surface Plasmon Resonance method applied for this work has therefore revealed to be an accurate and highly sensitive strategy – with multiplexing possibility - for the sensing and detecting of different kind of biomolecules, from DNA fragments to whole bacteria cell.
Il mio lavoro di Tesi di Dottorato, sviluppato presso i laboratori Veneto Nanotech (Nanofab a Marghera, LaNN a Padova ed ECSIN a Rovigo), ha avuto come obiettivo l’utilizzo della tecnologia di risonanza plasmonica di superficie (SPR – Surface Plasmon Resonance) per lo sviluppo di piattaforme biosensoristiche per applicazioni clinica ed ambientali. In particolare, durante il lavoro di Dottorato sono state messe a punto due piattaforme SPR: la prima piattaforma utilizza sonde oligonucleotidiche a DNA per l'individuazione di mutazioni causanti fibrosi cistica (CF) mentre la seconda utilizza anticorpi per il rilevamento di cellule di Legionella pneumophila. Entrambi i sensori sono basati sulla stessa strategia di rilevamento, ovvero l’utilizzo di una metodologia Grating Coupled – Surface Plasmon Resonance (GC-SPR) progettata utilizzando una configurazione conica di illuminazione ad azimut rotato per la rilevazione diretta – senza passaggi di marcatura, label-free – dell’analita in esame. Per quanto riguarda la piattaforma a DNA per la fibrosi cistica, si è sviluppata una strategia per l'individuazione di alcune delle mutazioni più frequenti responsabili CF tra la popolazione italiana. Per la rilevazione di tali mutazioni le superfici di analisi utilizzate sono grigliati sinusoidali, e la rilevazione specifica delle sequenze di interesse si ottiene attraverso l'utilizzo di oligonucleotidi allele-specifici (ASO – allele specific oligonucleotide) con struttura ad hairpin, disegnati per la discriminazione di un singolo nucleotide. I substrati plasmonici sono stati utilizzati per testare campioni wild-type ed eterozigoti (wt/mut) per le mutazioni in esame, amplificati tramite PCR a partire da campioni clinici. Le condizioni di ibridazione sono state ottimizzate per ottenere il rapporto di discriminazione (DR – discrimination ratio) massimo tra campioni wild-type ed eterozigoti. I segnali SPR ottenuti ibridando campioni wild-type e campioni eterozigoti hanno mostrato DR in grado di identificare univocamente i genotipi corretti, come confermato da esperimenti di fluorescenza in microarray eseguiti in parallelo. Inoltre la genotipizzazione ottenuta tramite SPR non è stata inficiata in campioni contenenti DNA interferente, consentendo quindi di utilizzare la piattaforma per la discriminazione in parallelo dei diversi alleli, e la possibilità futura di scalare il sistema con un approccio di high throughput screening. Per quanto riguarda la piattaforma ad anticorpi per la rilevazione di Legionella pneumophila, la medesima strategia basata su GC-SPR è stata messa a punto per ottenere una rilevazione rapida e sensibile di tale patogeno. Il limite legale di L. pneumophila in ambienti ospedalieri ad alto rischio in Italia è di 102 UFC/L (unità formanti colonia) e la metodologia di riferimento per la sua identificazione è una tecnica di coltura microbiologica che richiede tempi di attesa fino a 7 giorni. Partendo da tali considerazioni un sistema GC-SPR altamente sensibile è stato sviluppato ed applicato per la rivelazione di L. pneumophila: la rivelazione è stata accuratamente impostata ed ottimizzata con un ceppo standard del battere, prima attraverso l'utilizzo di superfici d’oro non nanostrutturate (flat) opportunamente funzionalizzate ed analizzate tramite fluorescenza, e successivamente attraverso reticoli sinusoidali (grating) d’oro analizzati tramite elissometria GC-SPR. Attraverso la metodologia GC-SPR ad azimut rotato è stato possibile rilevare fino a 10 UFC, mentre con l’analisi in fluorescenza non è stato possibile identificare quantitativi di battere inferiori a 104 UFC. Risultati positivi sono stati ottenuti anche incubando campioni di L. pneumophila isolati direttamente dall’ambiente ospedaliero. Questa piattaforma di rilevazione potrà essere implementata come prototipo in cui campioni di acqua e aria potranno venir sequenzialmente concentrati, iniettati in un sistema di microfluidica, ed incubati sulla superficie del sensore SPR per l'analisi, obiettivi questi del progetto POSEIDON (Horizon2020) attualmente in corso. La particolare metodologia GC-SPR ad azimut rotato applicata in questo lavoro di Tesi si è dimostrata essere una strategia accurata e altamente sensibile - con possibilità di multiplexing - per la rilevazione di diversi tipi di biomolecole, a partire da frammenti di DNA fino ad intere cellule batteriche.

Condizioni meteorologiche e clima influenzano fortemente il settore delle produzioni animali. Lo stress da caldo determina una significativa riduzione dell'attività metabolica, della produzione, della capacità riproduttiva e una maggiore predisposizione alle malattie. Nel contesto biologico, la temperatura dell’aria è sicuramente considerata il principale fattore di stress, tuttavia, un’elevata umidità peggiora l'effetto della temperatura riducendo le perdite di calore per evaporazione e ostacolando quindi l’eliminazione del calore in eccesso. Scopi principali della ricerca sono stati la caratterizzazione dell’area del Mediterraneo in termini di Temperature Humidity Index (THI) e stabilire le relazioni tra stagione/THI e mortalità nella bovina da latte allevata in un’area geografica Italiana altamente vocata per questo tipo di allevamento. I risultati suggeriscono che, allevatori e politici che operano nell’area Mediterranea, dovrebbero tenere nella dovuta considerazione variabilità e scenari del THI nella pianificazione degli investimenti nel settore delle produzioni animali. Conoscere in anticipo il verificarsi di condizioni climatiche avverse permetterebbe agli allevatori di metter in atto misure di contrasto sugli effetti negativi delle condizioni climatiche. Inoltre, dovrebbero venire sviluppate misure di adattamento appropriate per contesti specifici in termini di cultura, società, o sistemi politici, che possano contribuire alla sostenibilità ambientale, nonché allo sviluppo economico e alla lotta alla povertà.
Weather and climate strongly influence the field of animal production. Heat stress causes a significant reduction in metabolic activity, production, reproductive capacity, and increases susceptibility to diseases. In the biological context, the air temperature is definitely considered the main factor of stress, however, high humidity worsens the effect of temperature by reducing the evaporation heat loss and thus preventing the removal of excess heat. The main purposes of the research were the characterization of the Mediterranean basin in terms of Temperature Humidity Index (THI) and to establish relationships between season/THI and mortality in dairy cattle bred in an Italian geographic area with a high concentration of dairy farms. THI variability and scenarios should be taken into careful consideration by farmers and policy makers operating in Mediterranean countries when planning investments. Investments should at least partially be directed to implementation of adaptation measures, which may support farmers in the transition to climate-smart agriculture and help them to alleviate the impact of hot extremes on animal welfare, performance and health. In addition, measures should be developed appropriate adaptation to the specific contexts in terms of culture, society, or political systems, which can contribute to environmental sustainability and economic development and fighting poverty.

Condizioni meteorologiche e clima influenzano fortemente il settore delle produzioni animali. Lo stress da caldo determina una significativa riduzione dell'attività metabolica, della produzione, della capacità riproduttiva e una maggiore predisposizione alle malattie. Nel contesto biologico, la temperatura dell’aria è sicuramente considerata il principale fattore di stress, tuttavia, un’elevata umidità peggiora l'effetto della temperatura riducendo le perdite di calore per evaporazione e ostacolando quindi l’eliminazione del calore in eccesso. Scopi principali della ricerca sono stati la caratterizzazione dell’area del Mediterraneo in termini di Temperature Humidity Index (THI) e stabilire le relazioni tra stagione/THI e mortalità nella bovina da latte allevata in un’area geografica Italiana altamente vocata per questo tipo di allevamento. I risultati suggeriscono che, allevatori e politici che operano nell’area Mediterranea, dovrebbero tenere nella dovuta considerazione variabilità e scenari del THI nella pianificazione degli investimenti nel settore delle produzioni animali. Conoscere in anticipo il verificarsi di condizioni climatiche avverse permetterebbe agli allevatori di metter in atto misure di contrasto sugli effetti negativi delle condizioni climatiche. Inoltre, dovrebbero venire sviluppate misure di adattamento appropriate per contesti specifici in termini di cultura, società, o sistemi politici, che possano contribuire alla sostenibilità ambientale, nonché allo sviluppo economico e alla lotta alla povertà.
Weather and climate strongly influence the field of animal production. Heat stress causes a significant reduction in metabolic activity, production, reproductive capacity, and increases susceptibility to diseases. In the biological context, the air temperature is definitely considered the main factor of stress, however, high humidity worsens the effect of temperature by reducing the evaporation heat loss and thus preventing the removal of excess heat. The main purposes of the research were the characterization of the Mediterranean basin in terms of Temperature Humidity Index (THI) and to establish relationships between season/THI and mortality in dairy cattle bred in an Italian geographic area with a high concentration of dairy farms. THI variability and scenarios should be taken into careful consideration by farmers and policy makers operating in Mediterranean countries when planning investments. Investments should at least partially be directed to implementation of adaptation measures, which may support farmers in the transition to climate-smart agriculture and help them to alleviate the impact of hot extremes on animal welfare, performance and health. In addition, measures should be developed appropriate adaptation to the specific contexts in terms of culture, society, or political systems, which can contribute to environmental sustainability and economic development and fighting poverty.

Tra le applicazioni all'avanguardia di materiali a base di carbonio nanostrutturato, come grafene e nanotubi di carbonio (CNTs), l'analisi del respiro (i.e. breathomics), il monitoraggio ambientale e l'industria alimentare stanno oggigiorno sfidando la fisica, la chimica e l’ingegneria dei materiali a sviluppare piattaforme di sensori estremamente sensibili, affidabili e stabili, che siano in grado di rilevare piccolissime quantità (ordine dei ppb) di molecole di gas nell’ambiente che le circonda. In questa tesi, verrà presentato lo sviluppo di 6 piattaforme di sensori di gas. Queste piattaforme saranno sviluppate con carbonio nanostrutturato e avranno come scopo principale quello di discriminare potenziali patologie attraverso il riconoscimento di pattern molecolari presenti nel respiro esalato, nonché la loro possibile applicazione nel monitoraggio ambientale degli inquinanti e nell’industria alimentare. Questo obbiettivo verrà realizzato sviluppando dapprima piattaforme a base di CNTs su un substrato di plastica o su silicio/ossido di silicio e successivamente a base di grafene su nitruro di silicio. Verranno esplorati diversi metodi di funzionalizzazione sia per i CNTs che per il grafene, per aumentarne la sensitività, e verranno utilizzati diversi materiali per la funzionalizzazione, incluse nanoparticelle, molecole organiche o sali di diazonio. Tecniche di spettroscopie Raman ed elettroniche unitamente a microscopia a forza atomica verranno utilizzare per caratterizzare i campioni, mentre le esposizioni di gas verranno effettuate in aria, condizione più simile a quella delle applicazioni finali dei sensori, cercando di indagare concentrazioni dei gas selezionati nel sub-ppm o di poche decine di ppm. L’analisi delle componenti principali (PCA) verrà utilizzata per testare le capacità di discriminazione dei gas delle piattaforme sviluppate. Infine, uno dei nasi elettronici sviluppati verrà testato con il respiro esalato di soggetti sani o affetti da broncopneumopatie cronico ostruttive (COPD), dimostrando un’ottima capacità di discriminare e riconoscere le due classi di pazienti.
Among forefront applications of nanostructured carbon materials such as graphene and nanotubes, breathomics, environmental monitoring and food industry are challenging physics, chemistry and device engineering to develop extremely sensitive, selective, and stable platform to recognize ppb amount of target molecules in the environment. In this thesis, the development of 6 platforms will be presented. The platforms are based on nanostructured carbon aimed mostly to discriminate potential pathologies through pattern recognition in molecular fingerprint of breath samples, but also for environmental monitoring or food industry applications. This objective will be realized through properly developed devices based first on CNTs on a plastic substrate or on silicon/silicon oxide substrate and then on graphene on silicon nitride. Different kinds of functionalization techniques of graphene and CNTs will be explored to enhance the sensitivity of the pristine layers, as well as different functionalization materials, going from nanoparticles to organic molecules or diazonium salt precursors. The characterization of the materials involves electron and Raman spectroscopies, as well as atomic force microscopy, while gas exposures are carried out in the lab environment, which is much closer to the destination of the developed sensors, trying to investigate a low-ppm range or sub-ppm range of the considered gases. The gas discrimination is assessed through principal component analysis (PCA). Finally, one of the developed devices is exposed to the exhaled breath of healthy subjects or patients affected by chronic obstructive pulmonary diseases (COPD), demonstrating a remarkable capability to discriminate between healthy and sick patients.

Milk and dairy products are an excellent source of bioavailable calcium thanks to the presence of casein phosphopeptides (CPPs), that with their phosporylated serines, bind calcium ions, keeping them in a soluble and bio-absorbable state. Digestion and absorption are limiting steps for the peptide bioavailability; thus the in vitro digestion procedures combined with the use of intestinal cell models allowed to mimic the physiological milieu, where digestion occurs and the interactions between peptides and intestine determine their fate. The purpose of this PhD thesis was to identify the biological potential of casein phosphopeptides after in vitro digestion. The first step was about the bio accessibility property of a CPP purified mixture using a culture of intestinal human cells. Next step was about the bioactivity study of a complete food containing CPPs, represented by Grana Padano (GP) and Trentin Grana (TN) at 13, 19 and 26 months of ripening. Present data show that after in vitro digestion, preformed aggregates (CPP + Ca) in a commercially mixture still maintain their conformation and retain their bioactivity as promoters of
calcium uptake in intestinal cells, regardless of calcium mechanism transport (TRPV6 in Caco2 cells and depolarizing L-type calcium channels in HT-29 cells). GP and TN digestates, were also able to induce a calcium uptake in a co-culture of Caco2 and HT-29, a model of human intestinal epithelium more complete and useful. Moreover, they shown the capacity to induce the bone mineralization in human osteoblast-like cells. Since the biological potential after digestion was verified, CPPs could be considered as a nutraceutical and GP and TN cheeses as potential functional foods.

Immunotherapy represents a therapeutic option for subgroups of paediatric patients with leukaemia who, despite the impressing advances of the last decades in the field, still show a poor prognosis because of high risk-disease or relapse. A deeper understanding of how the immune system physiologically recognizes and eradicates tumour cells is mandatory. Peptidic antigens are of great interest in the field of immunotherapy because they could be used as vaccines to boost immunity. TEL/AML1 mutant protein, whose sequence is known, is the result of a balanced t(12;21) translocation which generates a fusion gene. Peptides can be artificially synthetized from TEL/AML1 fusion protein and their HLA-binding capacity and immunogenicity can be predicted through bioinformatic tools. This project aimed to investigate whether the excellent prognosis showed by patients who suffer from a B-lineage ALL harbouring the TEL/AML1 mutation could be related to an immune response against peptidic antigens derived from the TEL/AML1 mutant protein. For such purpose, 8 priming experiments were performed with healthy donors’ leucocytes. Six experiments were carried out according to a dendritic cells-mediated protocol, whereas two experiments were performed according to a beads-mediated protocol. Successfully primed lymphocytes (identified by mean of intracellular cytokines production) were selected through flow cytometric sorting and single-cell seeded in order to get T-cell clones. This was possible in 3 out of 8 priming experiments. Growing T-cell clones were tested after stimulation with peptides (or through tetramer staining) but they did not show enough specificity. We also tried to show an immune response against fusion peptides in peripheral blood leucocytes of patients who survived a TEL/AML1 positive B-lineage ALL, through exposure to peptides and a short course stimulation with cytokines. We tested 22 patients, but unfortunately we weren’t able to show an answer against fusion peptides in any of them. Possible reasons might be the lack of specificity of the activation markers we used to identify reactive cells, the not enough restrictive gates we used for sorting, the fact that the HLA super type B*07 (for which the restricted peptides had the best prediction score) was underrepresented in our patients’ cohort. We suggest to perform further experiments using new activation markers, such as CD25 or PD-L1, or different techniques to identify reactive cells (such as Elispot), to use more restrictive gates for sorting and to exploit the beads priming protocol. In order to sample such lymphocyte populations (i.e. antigen specific T-cells) with an extremely low frequency, a possibility may be collect repeatedly blood samples from the same patient at different time points. Further studies are warranted to test the hypothesis of an autologous, spontaneously arising immune response against TEL/AML1 fusion peptides as reason for the good prognosis of TEL/AML1 positive leukaemia. Another possible approach in order to validate fusion peptides might be to test them in a situation of HLA B*07 mismatch between lymphocytes and APCs. The clinical counterpart could be the generation of reactive T-cell clones, cloning of their TCR and its transduction in the patient’s or donor’s lymphocytes, the latter in the perspective of a post-hematopoietic stem cell transplantation adoptive immunotherapy.
L’immunoterapia costituisce un’opzione terapeutica per alcuni sottogruppi di pazienti con leucemia dell’età pediatrica i quali, nonostante i notevoli progressi degli ultimi decenni, ancora non mostrano una prognosi soddisfacente perché affetti da malattia ad alto rischio oppure da ricaduta. Una comprensione più profonda di come il sistema immunitario fisiologicamente riconosce ed elimina le cellule tumorali è essenziale. Gli antigeni peptidici sono di grande interesse nel settore dell’immunoterapia perché possono essere utilizzati come vaccini per potenziare l’immunità. La proteina mutante TEL/AML1, la cui sequenza è nota, è il risultato di una traslocazione bilanciata t(12;21) che genera un gene di fusione. Dalla proteina di fusione TEL/AML1 si possono sintetizzare artificialmente peptidi, la cui capacità di legare le molecole HLA ed immunogenicità si può prevedere attraverso strumenti bioinformatici. Questo progetto ha l’obiettivo di indagare se l’eccellente prognosi dei pazienti affetti da leucemia linfoblastica di linea B con la mutazione TEL/AML1 possa essere correlata ad una risposta immunologica nei confronti di peptide di fusion derivati dalla proteina mutante TEL/AML1. A tale scopo, sono stati realizzati 8 esperimenti di priming con leucociti di donatori sani. Sei sono stati realizzati secondo un protocollo mediato da cellule dendritiche, mentre altri due esperimenti sono stati condotti secondo un protocollo mediato da beads. I linfociti responsivi al processo di priming (identificati mediante la produzione intracellulare di citochine) sono stati selezionati mediante sorting citofluorimetrico e coltivati a singola cellula in modo da ottenete cloni T-cellulari. Ciò è stato possibile in 3 esprimenti su 8. I cloni T-cellulari con evidenza di crescita sono stati testati dopo re-stimolazione con i peptidi (o mediante tetramer-staining) ma non hanno dimostrato sufficiente specificità- Abbiamo inoltre provato a dimostrare una risposta immunologica nei confronti dei peptidi di fusione nei leucociti (da sangue periferico) di pazienti con leucemia linfoblastica di linea B TEL/AML1 positiva in remissione, mediante esposizione ai peptidi e una breve stimolazione con citochine. Sono stati testati 22 pazienti, ma purtroppo non è stato possibile evidenziare una risposta nei confronti dei peptidi di fusione in nessuno di loro. Possibili spiegazioni potrebbero essere la mancanza di specificità dei marcatori di attivazione che sono stati utilizzati per identificare le cellule reattive, i gate non sufficientemente restrittivi utilizzati per il sorting, il fatto che il supertipo HLA B*07 (i peptidi B*07 ristretti avevano il migliore score predittivo) era sotto-rappresentato nella coorte di pazienti presa in esame. Ci riproponiamo di realizzare ulteriori esperimenti utilizzando nuovi marcatori di attivazione, come CD25 o PD-L1, oppure differenti tecniche per identificare le cellule reattive (come l’Elispot), di usare gates più restrittivi per il sorting e di utilizzare esclusivamente il protocollo mediato da beads per il priming. Per riuscire a includere nel campione popolazioni linfocitarie (cellule T antigene-specifiche) la cui frequenza è estremamente bassa, una possibilità potrebbe essere eseguire prelievi ematici ripetuti nel tempo nello stesso paziente. Sono necessari ulteriori studi per testare l’ipotesi di una risposta immune autologa, spontanea, nei confronti dei peptidi di fusione TEL/AML1 come spiegazione della buona prognosi della leucemia linfoblastica di linea B TEL/AML1 positiva. Un altro possibile approccio per validare i peptidi di fusione potrebbe essere quello di testarli in una situazione di HLA B*07 mismatch tra linfociti ed APCs. La ricaduta clinica potrebbe essere la generazione di cloni T-cellulari dalle cellule reattive al priming, il clonaggio del loro TCR e la sua transduzione nei linfociti del paziente o del suo donatore, in quest’ultimo caso nella prospettiva di un’immunoterapia adottiva post-trapianto di cellule staminali ematopoietiche.

L’évolution et l’utilisation croissante des systèmes de communications mobiles sont associées à des études en laboratoire pour s’assurer de l’innocuité d’une exposition aux ondes électromagnétiques radiofréquences. Dans ce contexte, ce mémoire se concentre sur la caractérisation de systèmes d'exposition permettant des études en laboratoire sur des modèles cellulaires in vitro. Une double approche, numérique et expérimentale, est mise en œuvre pour réaliser la dosimétrie de ces dispositifs, afin de déterminer et maîtriser les niveaux d’exposition. Une des problématiques liées à cette dosimétrie est due aux dimensions micrométriques mises en jeu. Aussi, une technique de microscopie basée sur un marqueur fluorescent dépendant de la température, nommé Rhodamine B, a été mise en place et évaluée. Il ressort de cette évaluation une recommandation sur la concentration du marqueur de l’ordre de 50 μM. Après calibration, il est alors possible d’estimer le débit d’absorption spécifique (DAS) à partir de la variation de température, et ce même pour de faibles niveaux de DAS (< W/kg) avec une résolution spatiale inférieure à la dizaine de micron. On parle alors de microdosimétrie. Les deux principaux systèmes d’exposition étudiés, basés sur des réseaux de microélectrodes (MEA), permettent l’enregistrement d’une activité électrophysiologie de neurones. L’exposition aux ondes électromagnétiques est réalisée simultanément en insérant ces MEA dans des cellules TEM. De la dosimétrie effectuée à 1.8GHz, il ressort une plus forte sensibilité du premier MEA à son environnement. On a montré que les modifications apportées au second MEA, taille de l’ouverture et plan de masse, ont permis de limiter de façon significative l’influence de l’environnement. La microdosimétrie a mis en évidence une bonne homogénéité du DAS entre les électrodes avec une valeur estimée à 7±1W/kg pour1W incident. Enfin, un dispositif d'exposition microfluidique basé sur un guide d'ondes coplanaire a été caractérisé en conditions statiques
The evolution and increasing use of mobile communications systems was associated with laboratory investigations to study radiofrequency electromagnetic waves exposure safety. In this context, this thesis focuses on the characterization of exposure systems allowinglaboratory in vitro studies on cell models. A dual numerical and experimental approach is implemented to perform the devices dosimetry allowing to determine and control the exposure levels. One of the limitations associated with this dosimetry is due to the micrometric dimensions involved. Therefore, a microscopy technique based on a temperature-dependent fluorescent dye named Rhodamine B was set up and evaluated. This assessment recommends an optimal concentration of the dye at around 50 μM. After calibration, it is possible to estimate the specific absorption rate (SAR) from the temperature variation, even for low levels of SAR (< W / kg) with a spatial resolution of less than ten micrometers i.e. microdosimetry. The two main exposure systems studied, based on microelectrode arrays (MEA), allow the recording of neurons electrophysiological activity. Exposure to electromagnetic waves is achieved simultaneously by inserting these MEAs into TEM cells exposure systems. Dosimetry carried out at 1.8 GHz shows a higher sensitivity of one MEA to its environment. It was shown that the modifications made to the second MEA such as its aperture size and ground planes, have reduced the proximity environment influence. The microdosimetry demonstrated good homogeneity of the SAR between the electrodes with an estimated value of 7 ± 1 W / kgfor 1 W incident power. Finally, a microfluidic exposure device based on a coplanar waveguide was characterized under static conditions

BACKGROUND Maternal obesity (MO) is expanding worldwide, contributing to Gestational Diabetes Mellitus (GDM) prevalence. The altered placental function and intrauterine environment of MO and GDM are associated with adverse maternal and fetal outcomes, with short- and long-term complications. Growing evidence suggests that both MO and GDM are characterized by placental autophagy disturbances, mitochondrial (mt) dysfunctions and epigenetic alterations, which could contribute to the pathogenesis of pregnancy-related metabolic diseases. For these reasons the aim of this work was to characterize placental autophagy and mitochondrial bioenergetics, and maternal plasmatic microRNAs profile in the context of MO and GDM. METHODS 60 women with spontaneous singleton pregnancies delivering by elective Caesarean section were enrolled: 25 normal-weight (NW), 22 obese without comorbidities (OB/GDM(-)), 13 obese with GDM (OB/GDM(+)). The expression of antioxidant metabolism markers and autophagy-related genes expression was evaluated in placental villous tissue by Real-time PCR with SYBR green chemistry. In placenta, we also quantified ATP by CellTiter-Glo Luminescent Cell Viability Assay adapted to placental villous tissue, and mt Complexes I-V protein content by Western Blot experiments. Moreover, in a subgroup of 7 NW, 6 OB/GDM(-) and 6 OB/GDM(+), microRNA (miRNA) profiling in maternal plasma at delivery was performed with miRCURY LNA Serum/Plasma miRNA Focus PCR Panel (Qiagen). Statistical analysis and bioinformatics predictive tools: SPSS v.27 (IBM); GeneGlobe (Qiagen); miTALOS v.2; miRPath v.3. RESULTS AND CONCLUSIONS Placental antioxidant genes expression (CAT, GPX1, GSS, GSR) tended to decrease, the pro-autophagic ULK1 increased and the chaperone-mediated autophagy (CMA) regulator PHLPP1 decreased in OB/GDM(-) vs NW. On the other hand, PHLPP1 expression increased in OB/GDM(+) vs OB/GDM(-). These preliminary findings showed placental alterations of autophagy in MO and GDM, paving the way for further analyses aimed at elucidating the role of placental autophagy in metabolic disorders during pregnancy. Moreover, placentas of OB/GDM(-) and OB/GDM(+) women had reduced ATP level vs NW. All OB showed decreased Complex I vs NW. Furthermore, lower expression of Complexes III and V was found in OB/GDM(+) group (vs NW; vs NW and OB/GDM(-), respectively), along with a reduced fetal/placental weight ratio (i.e. placental efficiency) vs NW. These results might suggest impaired placental mt function in obese subjects, especially with GDM. Sexual dimorphism in terms of the above-mentioned placental molecular markers was also investigated, with intriguing results suggesting that females and males might respond differently to insults and/or have differential, sex-dependent compensatory mechanisms in order to deal with the perturbed environment. MiRNA profiling in maternal plasma highlighted patterns of significantly differentially expressed miRNAs between the three groups. In particular, OB/GDM(-) vs NW differed in the expression of 4 miRNAs, OB/GDM(+) vs NW of 1, and OB/GDM(+) vs OB/GDM(-) of 14. Pathway enrichment analysis by miRPath and miTALOS revealed many pathways associated with the set of miRNAs of each comparison, among which the most interesting and relevant in our context were related to nutrients and hormones metabolism, inflammation and oxidative stress. Indeed, miRNAs could be promising biomarkers of metabolic alterations in MO and GDM. Nevertheless, future investigations are needed to further deepen the pregnancy epigenetic landscape.

Scopo della ricerca sviluppata durante questi tre anni di Dottorato è quello di spiegare i meccanismi sottesi alla risposta allo stress ossidativo nell'uomo a livello integrativo e molecolare. Per questo fine, nel primo anno di Dottorato, si è innanzi tutto implementato un metodo quantitativo assoluto atto a valutare la produzione dei radicali liberi. Come è noto ciò che importa è l’equilibrio tra produzione di Specie Reattive dell’Ossigeno (ROS) e la disponibilità di difese antiossidanti. Quando questo equilibrio viene meno, si parla di Stress Ossidativo, con conseguenze a carico degli acidi nucleici, dei lipidi e delle proteine, compromettendo il metabolismo e la vitalità cellulare, fino a indurre apoptosi o necrosi. Questo è un fenomeno ricorrente in molte malattie acute e croniche oltre che nel fisiologico processo di invecchiamento. Allo scopo si è utilizzata la tecnica di Risonanza Paramagnetica Elettronica (EPR) accoppiata a specifiche sonde molecolari (spin trapping) per sviluppare un metodo quantitativo “in vivo” in grado di monitorare la produzione di ROS in campioni biologici quale il sangue e sue frazioni. Gli inizi del mio periodo di Dottorato sono fortunatamente coincisi con l’acquisizione da parte dell’Istituto di Bioimmagini e Fisiologia Molecolare (IBFM) del CNR (Lita di Segrate), di uno strumento EPR che risponde alle esigenze di maneggevolezza e portabilità (e-scan Bruker BioSpin, Germania), di recente immissione sul mercato, che opera in onda continua (CW), nella banda X delle µW, in grado di rilevare concentrazioni molto basse (nM) in piccoli campioni (50 µl), utilizzando una cavità risonante le cui proprietà ottimizzano il fattore di riempimento, conferendo un’elevata sensibilità allo strumento. L’attendibilità dell'efficacia del metodo sviluppato è stata verificata con correlazioni tra metodi classici enzimatici e misure EPR in vari stati: fisiologici e parafisiologici. Trattandosi della messa a punto di un metodo di misura completamente innovativo e mancando pertanto dati di letteratura cui fare riferimento, si sono rese necessarie molte prove sperimentali da campioni ematici, in un vasto gruppo di soggetti, con tantissime acquisizioni modificando e ottimizzando di volta in volta i parametri e le condizioni (T, pO2) Questo procedimento è stato lento e laborioso; una volta superate le difficoltà tecniche, si è avuto a disposizione un piccolo catalogo di misurazioni che è servito ad ottenere i primi dati sperimentali che saranno illustrati nei primi paragrafi di questa tesi. Si è inoltre cercata una correlazione statistica tra la produzione di ROS determinata con tecnica EPR ed i marcatori dello stress ossidativo ottenuti dai vari test convenzionali enzimatici riportati in letteratura quali il dosaggio da sostanze reattive all'acido tiobarbiturico (TBARS) e carbonili proteici (PC) in grado di quantizzare il danno “a posteriori” prodotto dai ROS su lipidi e proteine rispettivamente. Una correlazione sistematica tra i dati EPR e i dati enzimatici non era certa, in quanto questi ultimi rivelano un danno conclamato. Tuttavia, è stata trovata una significativa correlazione (ANOVA, p<0.05) in soggetti a riposo, tra la produzione di ROS/TBARS e PC (rispettivamente: r2=0.74 e r2=0.60). Per rispondere allo scopo della ricerca, abbiamo assunto come modello l’ipossia studiata 1) come situazione che induce uno sbilanciamento tra Stress Ossidativo e capacità antiossidante e 2) nel tentativo di identificare i possibili meccanismi di ripristino dell’equilibrio. Dall'insieme dei dati raccolti, al di là dei risultati specifici, possiamo evincere che anche se la misura quantitativa assoluta della produzione di ROS (umol . mim-1) non può essere un parametro generalizzato, perché funzione di variabili fisico-chimiche (pO2, Temperatura, concentrazione della sonda), acquisizione ed elaborazione dei parametri vanno comunque prese in considerazione. Dal calcolo dei limiti di detenzione (LOD): 30 · 10-3 mM, e quantificazione (LOQ): 100 · 10-3 mM, lo strumento si è rilevato sensibile e affidabile. Allo stesso tempo, va tenuto presente che il dato ottenuto rappresenta la produzione di ROS non tamponati dal sistema antiossidante. Per questo, si è ritenuto che la misura su campione ematico fosse rappresentativa della risposta sistemica integrativa. In particolare, il sangue capillare, oltre alla sua elevata sensibilità e riproducibilità (0,5% di differenza tra misure ripetute), ci ha permesso di disegnare un protocollo agile mini-invasivo. Da quanto fin qui esposto, possiamo ricavare che, al di là della ricerca di un dato assoluto da riportare in letteratura, è una valida procedura che ogni soggetto, monitorato nel tempo risulti controllo di se stesso, ovvero che a indice della risposta equilibrio/squilibrio vengano quantizzate le variazioni. Nel tentativo di identificare i possibili meccanismi di ripristino dell’equilibrio dello stress ossidativo, gli esperimenti sono stati condotti su: A) soggetti sani, che come tali sono caratterizzati da una condizione di equilibrio tra ROS e difese antiossidanti, B) soggetti affetti da patologie neurodegenerative, per i quali era logico attendersi, come confermato dalla realtà, che fossero caratterizzati da una condizione di conclamato squilibrio, rivelabile da un significativo incremento di ROS prodotti in condizioni di riposo. In soggetti sani, si sono disegnati, messi a punto e applicati differenti protocolli sperimentali, per testare l’effetto: a) dell’esercizio fisico (stato ipossico transitorio) di breve (hockeisti e nuotatori) e lunga durata (atleti di triathlon); b) dell’allenamento (nuotatori); c) dell’ipossia normobarica e ipobarica acuta e prolungata (giovani soggetti sedentari); d) dell’assunzione di molecole antiossidanti sulla produzione di ROS. In patologie neurodegenerative (pazienti affetti da: Neuropatia Diabetica (tipo II DN), decadimento cognitivo lieve (MCI) e Sclerosi Laterale Amiotrofica sporadica (sSLA)) dopo aver rilevato uno squilibrio in condizioni basali, è stato studiato l’effetto dell’esercizio/allenamento (SLA) o dell’assunzione di antiossidanti (tipo II DN) sulla possibilità di un potenziale ripristino a valori di equilibrio. Importanti risultati sono emersi dall’analisi dei dati registrati nelle varie condizioni sperimentali e sono illustrati in questa tesi. ll tema comune di tutti i test, oltre all’evidente studio sulla produzione dei ROS, è stato quello dell’ipossia. Infatti l’Ipossia, nelle sue varie espressioni (intese come deficienze parziali o totali di O2 nel nostro organismo), è stato elemento caratterizzante in tutti i protocolli che abbiamo testato. I protocolli di studio su pazienti sono tuttora in corso; pertanto i dati illustrati devono intendersi come preliminari. Nei soggetti patologici si è costatata una differente produzione di ROS in condizioni di riposo, mostrando un valore significativamente (p<0,05) più alto nei pazienti affetti da SLA (+20%) rispetto ai soggetti sani. L’allenamento, in questi pazienti, sembra giocare un ruolo favorevole nel diminuire la produzione di ROS: un decremento del 7% è stato calcolato nei valori a riposo pre e post allenamento. Un sicuro effetto positivo dell’allenamento controllato, dopo otto settimane, è stato osservato nei nuotatori (-25%) sulla produzione di ROS (2.24 ± 0.14 μmol · min-1). Tutti questi esperimenti sono stati condotti e i risultati ottenuti durante il secondo e terzo anno della mia Scuola di Dottorato. A parte gli specifici risultati ottenuti dal set di dati raccolti, osservazioni generali possono essere riassunte come segue: 1) Non vi è alcun modo per ottenere dati per quantificare il volume totale di ROS prodotta da un soggetto in qualsiasi condizione. Il dato EPR ricavato dalle misurazioni rappresenta l’eccesso di ROS che il sistema ha prodotto (μmol · min-1) in una determinata condizione, ed è quindi un tasso di produzione di ROS Inoltre si è scelto di effettuare la misura da sangue invece che da campioni di tessuto in modo da ottenere dati il più possibile sistematici. In particolare, il sangue capillare (rispetto al sangue venoso o plasma) ha dato la possibilità di sviluppare un metodo di misurazione affidabile, relativamente semplice e mini-invasivo. Tuttavia quando si passa da misurazioni di sangue capillare a venoso, bisogna prendere in considerazione la differente pO2. (la produzione di ROS in sangue venoso è stata trovata circa il 18% inferiore a quella nel sangue capillare: 1.79 ± 0.12 vs 1.48 ± 0.29 μmol·min-1 rispettivamente). 2) I dati non possono essere presi come parametri generali, poiché dipendono strettamente da variabili fisico-chimiche (T, pO2) Da tutti questi punti, si può evincere che, al di là della ricerca di un dato assoluto da riportare in letteratura, è una valida procedura che ogni soggetto, monitorato nel tempo risulti controllo di se stesso, ovvero che a indice della risposta equilibrio/squilibrio vengano quantizzate le variazioni. 3) Tuttavia si è consapevoli che il dato fornito da EPR deve essere integrato con dati sistemici quali: analisi ematochimiche di laboratorio (ematocrito, ferro, colesterolo, piastrine, neutrofili, linfociti, monociti), saturazione dell’ossigeno, cinetica degli scambi gassosi (VO2max). Si ritiene che il metodo possa trovare applicazione i) medicina dello sport offrendo un valido aiuto nei centri sportivi per monitorare l’efficacia dell’allenamento e gli eventuali effetti dello stress provocato ed eventualmente migliorare i protocolli; ii) alla medicina di montagna per lo studio delle patologie da alta quota, poiché l’ipossia è il primo e più delicato problema dell’altitudine sin dalla quota media (1800m); iii) ai centri clinici/diagnostici per monitorare gli effetti delle terapie/trattamenti sulla progressione di patologia. Seconda parte della mia attività di ricerca, è stato lo studio in vitro dell’ipossia da un punto di vista molecolare mediante misurazioni spettroscopiche EPR. Partendo da una visione molecolare, l’ipossia orchestra una moltitudine di processi e di pathway molecolari. Nel tradizionale meccanismo dell’ipossia si ha la presenza di una proteina eme in grado di rilevare il legame reversibile di O2 il quale causa un cambiamento allosterico nell’emo-proteina: da inattivo (forma ossigenata) ad attivo (forma de-ossigenata). Quindi l'attività di una proteina emo è determinata dalla presenza o assenza dell’ossigeno legato. Nella presente ricerca, la rilevanza di stati ipossici, dal punto di vista molecolare, atti ad essere rilevati sia in condizioni fisiologiche che patologiche è stato attribuito all’Ossido Nitrico (NO) e al ruolo della Mioglobina (Mb). E’ noto, infatti, che l’NO è un radicale libero particolarmente espresso in condizioni ipossiche in quanto è in grado di regolare la vasodilatazione, quindi aumenta l’afflusso di sangue e al tempo stesso può essere però dannoso andando ad inibire la catena respiratoria a livello del complesso IV. In letteratura sono riportate cinque isoforme di Mb umana (Mb I-V) presenti nel muscolo scheletrico in percentuali diverse (Mb I: 75%; Mb II: 20%; Mb III-V: 5%) le cui funzioni non sono ancora state chiarite. Prendendo a riferimento Mb II, queste proteine differiscono solo per un singolo residuo aminoacidico: E54K (Mb I), K133N(Mb III), R139Q (Mb IV) a R139W (Mb V). Inoltre popolazioni tibetane, native dell’alta quota, presentano come risposta all’ipossia alte capacità antiossidanti, alto flusso ematico sistemico, metabolismo ed efficienza meccanica migliore, alti livelli di metaboliti circolanti biologicamente attivi all’NO e sono caratterizzate da un significativo aumento della concentrazione di Mb a livello muscolare con particolare riferimento all’ isoforma 54E (Mb II). Il presente studio, grazie all’analisi combinata EPR e simulazioni di Dinamica Molecolare (MD), contribuisce ulteriormente a sottolineare il ruolo importante svolto dalla Mb in condizioni di ipossia; inoltre segna un primo passo verso l'identificazione dei diversi ruoli svolti dalle isoforme della Mb Importanti risultati funzionali si sono ottenuti dallo studio EPR: l’NO ha capacità di legame significativamente maggiore con l’isoforma 54E rispetto all’isoforma 54K; questa è più circoscritta alla struttura dello stato de-ossigenato e la capacità di legame diventa significativamente inferiore (p<0.01) e quasi identica per le due proteine in presenza di O2 anche a differenti livelli di pO2 (40 e 100 mmHg). Concludendo, osservazioni e prospettive future possono essere condensate nella convinzione, che confidiamo saranno condivise e verificate anche dal lettore, della validità del nuovo del metodo sviluppato e dei dati raccolti. Alcuni risultati, in particolar modo quelli concernenti gli stati patologici, sono da considerarsi preliminari; tuttavia la strada da percorrere è estremamente interessante e richiede ulteriori indagini.
The aim of the research developed in the time course of the three years of my Doctoral course in Molecular Medicine was to shed light into the mechanisms involved in Oxidative Stress responses in man at both integrative and molecular levels. To reach this general and high-level purpose we firstly focused our attention towards developing a method capable of returning an absolute quantitative estimation of Free Radicals production level. This particular aim was the subject of the first year of my researching activity. As a matter of fact, as is well known, in any physiological and/or pathological condition a subject could be involved, ‘oxidative stress’ results from the imbalance between Reactive Oxygen Species (ROS) production and his antioxidant capacity. Electron Paramagnetic Technique (EPR) resulted the method of choice since, as is well known, it is the only technique capable of returning, in a non-invasive way, the ‘intrinsic’ quantitative information (the signal is proportional to the number of excited electron spins). Indeed the beginning of my Doctoral period fortunately coincided with the acquisition by the Molecular Bioimaging and Physiology (IBFM) Institute of CNR (Lita Segrate) of an EPR instrument (e-scan Bruker BioSpin, Germany) of recent design, responding to the innovative characteristics of easy portability and handling. The spectrometer operates at the common X-Band microwave frequency and deals with very low concentration levels (nM) even in small sample volumes (50 μl). Aware of the novelty of the method, we first looked for a correlation between the attained EPR experimental data and those returned when adopting the classical enzymatic methods (e.g. ThioBarbituric Acid Reactive Substances (TBARS) and Protein Carbonyls (PC) data collected from the same subject). Indeed, a correlation could not, by principle, be expected, since, compared to the EPR data, enzymatic methods give a quantitative “a posteriori” evaluation of the damage produced by ROS, on lipids and cellular proteins. As a matter of fact, a positive correlation (ANOVA, p<0.05) was found between EPR ROS production data and TBARS (r2 ~ 0.7) as well as PC concentration (r2 ~ 0.6), which was found in resting subjects. Then, in order to achieve the overall objective of the research, we assumed ‘hypoxia’ as the ‘particular’ condition to be investigated, since it was viewed as a peculiar state capable of: 1) producing un imbalance between oxidative stress and anti-oxidant capacity; 2) offering the possibility of the identification of mechanisms involved in the perturbed balance restoring. To attain this ‘secondary’ aim, the experiments were carried out on: A) healthy subjects, that as such, were expected to start from a balance equilibrium condition; B) subjects affected by neurodegenerative pathologies, for whom, on the contrary, a sure imbalance condition was expected, that would be confirmed by a significant increment of ROS production, even at rest. However a fully innovative method had been developed, so that we lacked of referring literature data: a series of preliminary measurements on a homogeneous group of healthy subjects had to be firstly carried out, not only to optimize the acquisition and environmental conditions (T, pO2 that have to be carefully defined since they greatly affect EPR data), but also to reach reliable resting physiological values and range of variability. A great reproducibility of the data (μmol . min-1) was found on blood capillary samples taken from the same healthy subject six hours apart (r² = 0.99, ~ 0.5% discrepancy) and was calculated; limit of detection (LOD) and quantification (LOQ) of 30 ∙ 10-3 mM and 100 ∙ 10-3 mM were calculated respectively. In healthy subjects, the experimental protocols were designed in order to study the effects of: a) exercise (as inducing a transitory hypoxic state) of both short (in hockey players, swimmers) and long duration (in triathletes); b) training (as inducing a balance restore, in swimmers); c) hypoxia, both hypo- and normobaric (to study the effect of the assumed model condition itself, in young sedentary subjects); d) antioxidant molecules administration as an external way helping to restore the balance. In neurodegenerative pathologies (patients affected by: type II Diabetic Neuropathy (type II DN), Mild Cognitive Impairment (MCI) and sporadic Amyotrophic Lateral Sclerosis (sALS)) after checking the expected unbalance throughout a significant increase of ROS production (sALS versus healthy subjects: +20%, p<0.05) under resting conditions, the effect of training on the possibility of approaching a normal ROS production level was investigated. The collected data thus far, that have to be nevertheless considered as preliminary results, of the training effect in sALS patients showed a beneficial effects; an decrease ROS production resting value (about -7%) was calculated after twelve weeks of training by the patients. in swimmers a significantly lower (about -25%) basal ROS production value (2.24 ± 0.14 μmol · min-1) after eight weeks training period was calculated. All these experiments were carried out and results obtained during my second and third year of my Doctoral School. Besides and beyond the specific results obtained from the collected data set, pointed out throughout my thesis, general observations can be summarized as follows: 1) There is no way to obtain data quantifying the total amount of ROS produced by a subject under any condition. Rather, EPR data return amount of ROS there were not scavenged by the antioxidant system. In other words, under a given condition, they represent the excess of the ROS produced in a given time, that is ROS production rate (μmol · min-1). Moreover we chose to carry out the measurement on blood instead of tissue samples to obtain most possible integrative data. In particular capillary blood (with respect to venous blood or plasma) gave us the opportunity of developing a mini-invasive, reliable and easy measurement method. Nevertheless when going from capillary to venous data, the different blood pO2 must be taken into account. (ROS production in venous was found about 18% lower than in capillary blood: e.g. 1.79 ± 0.12 vs 1.48 ± 0.29 µmol · min-1 respectively). 2) The obtained data cannot be taken as general parameters, since they strictly depend on the physic-chemical variables (T, pO2) assumed for the measurement that must be taken into great account. From all these considerations, we can therefore conclude that beyond the possibility of obtaining absolute data to report for the first time in literature, this method was found as quite a sensitive and reliable mean to monitor the changes of each subject during time progression, therefore making each subject the control. 3) Despite the reliability and high reproducibility of the afore-developed method, we are anyway aware that EPR data have to be compared and integrated by collecting systemic data: hematochemical parameters (e.g. hematocrit, iron, cholesterol, platelets, neutrophils, lymphocytes, monocytes), oxygen saturation, maximal oxygen consumption (VO2max). Finally we are confident that our method will be in the near future considered suitable to be applied into different medical fields: i) sport medicine, as an effective available help in sports centers to set up and adjusts training protocols by monitoring training effects on oxidative stress. ii) mountain medicine, in studying pathologies induced by hypoxia following high altitude acute and chronic staying; iii) clinic and diagnostic centers, helping in monitoring progression of pathologies and/or effects of therapies. On the other side, second part of my researching activity, the study of hypoxia throughout EPR measurements, was carried out in vitro at a molecular level. In fact, hypoxia orchestrates a multitude of processes of molecular pathway responses. Nevertheless the traditional mechanism of sensing hypoxia involves the presence of a heme protein so that hypoxia can be detected by a reversible binding of O2 at the heme site, which causes an allosteric shift in the hemoprotein from inactive (oxyform) to active (deoxy) form. The activity of a hemoprotein is therefore determined by the presence or absence of bound oxygen. Among heme proteins, the relevance of hypoxic states, suitable to be encountered in both physiological and pathological conditions was, in the present research, made in conjunction with the supporting role recently attributed to deoxy-Myoglobin (Mb) as a ROS scavenger, with particular attention to a peculiar reactive species molecule, that is nitric oxide (NO). NO is a highly diffusive and reactive molecule, produced, in the cells, by the NO synthases enzymes (NOS), using L-arginine and O2 as substrates. Under normoxic conditions, if NO concentration becomes too high, oxy-Mb is able to scavenge and oxidize it into NO3-, maintaining mitochondrial O2 consumption, thus cellular respiration, at the optimum. On the contrary, under hypoxia, the low O2 availability leads to NOS inactivation (being substrate limited). The NO produced by Mb down-regulates mitochondria O2 consumption, contributing to elongate the intracellular oxygen gradient and limiting the formation of deleterious reactive oxygen species. In addition, Mb-produced NO might contribute to hypoxic vasodilatation. Thus, Mb’s nitrite reductase activity (very low pO2 level) might participate both in the modulation of tissue mitochondrial respiration and in the limitation of ischemia-reperfusion injury, under hypoxic. The relevance of hypoxic states, in conjunction with the supporting role recently attributed to deoxy-Myoglobin throughout its scavenging and vasodilatory properties was the rational basis for this study. As regard as Mb, in human’s skeletal muscle, up to five different Mb isoforms are reported in the literature (Mb I-V). Also different Mb percentages are present in the muscle: Mb I 75%, Mb II 29%, Mb III-V 5%. Taking Mb-II as a reference, these proteins differ only for one external amino acid and, namely, they can be seen as the E54K (Mb I), K133N (Mb III), R139Q (Mb IV) and R139W (Mb V) mutants, whose specific functions, if any, are completely unknown. In addition, these general observations were supported by specific findings on populations born and living in extreme hypoxic conditions possibly explaining their better metabolic and mechanic efficiency by the increase of Mb concentration, with particular reference to the 54E isoform (Mb II). Throughout a combined EPR and MD simulations analysis, the present study aimed to give experimental evidenced able to further underline the relevant role played by Mb under hypoxic conditions, then marking a first step towards the identification of different roles played by Mb isoforms, whose presence, among mammals, is a distinct feature of human Mb. Functional results obtained by the EPR study showed up not only a significantly greater NO binding capacity (more than double; p<0.01) of the 54E with respect to the 54K isoform was calculated from the EPR spectra integrated areas but even more circumscribed this property to the deoxygenate state, the binding capacity becoming significantly lower and almost the same for the two proteins in the presence of O2 at the two analyzed pO2 levels (40 and 100 mmHg). This novel and quite intriguing result was supported by structural considerations emerged by the MD simulation analysis and in turn mutually supported by structural EPR results. Concluding remarks and future perspectives can be summarized in our firm belief, which we are confident will be verified and accorded by the reader as well, of the novelty of the developed method and of the reported data. Indeed some of them, especially when concerning pathological subjects, have to considered ‘in fieri’, nevertheless the road to take is extremely interesting, and calls for further investigations.

Background Regenerative dentistry is a novel speciality in dentistry that aims to regenerate the loss tissue. It involves deep understanding of cell and molecular biology to design dental therapies that aim to restore, repair, rejuvenate, and regenerate dental tissues. Its application in oral and maxillofacial regeneration procedures namely; alveolar ridge preservation, periodontal regeneration, maxillary sinus augmentation, soft tissue augmentation, root coverage procedures to name a few is wide and has become common through the use of wide range of stem cells, biomaterials and biologics. The question remains unanswered because of few clinical trials available for each procedure to regenerate loss tissue. There is a strong need of evidence for such therapies for specific clinical indications in oral and maxillofacial regeneration. Since traditional meta-analysis compares only two types of intervention, network meta-analysis has been used as most common method to compare multiple interventions from different clinical trials. Therefore, using this methodology our main aim was to determine the best performing biomaterials and biologics for oral and maxillofacial regeneration procedures. The specific objectives were; 1. to determine the most effective grafting/sealing biomaterial in maintaining horizontal and vertical dimensions after alveolar ridge preservation; 2. to determine the most effective grafting/sealing biomaterial for new bone formation after alveolar ridge preservation; 3. to determine the most effective biomaterial for soft tissue regeneration after tooth extraction; 4. to determine the most effective biomaterial for gingival recession treatment in adjunct to coronally advanced flap; 5. to determine the most effective dental implant abutment material; 6. to rank the different combinations of recombinant human-derived growth and differentiation factors with/without scaffold biomaterial in the treatment of periodontal intrabony defects, through network meta-analysis of pre-clinical studies; 7. to determine the best rank recombinant growth factor formulations agents through network meta-analysis of clinical studies; 8. to determine the effectiveness of biomaterials used in soft tissue augmentation procedures; 9. to determine the most effective biomaterial in increasing residual bone height after maxillary sinus lift procedures Methods The study protocols were recorded in the PROSPERO database, and a standard approach to searching for articles in various scientific databases was adopted. A patient/population, intervention, comparison and outcomes (PICO) format was used to form a research question for each specific objective in oral and maxillofacial regeneration procedures. A custom search strategy was developed for each set of objectives and specific outcomes that accurately represented the successful results of specific oral and maxillofacial regeneration procedures were chosen. The mean, standard deviation, type of interventions used in each treatment group, number of participants and blinding status were extracted. The risk of bias was evaluated for the studies included in the network meta-analysis. The analysis was conducted using STATA software and the methods recommended by Chaimani A and Salanti G. The findings were presented in the form of network plots, inconsistency plots, predictive intervals, SUCRA rankings, and multi-dimensional scale rankings based on feasibility. The ranking of biomaterials and biologics was calculated for each considered outcome, and a Bayesian method was used in the methodology. A Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) checklist was followed to report the results of NMA. Results Specific objective 1: In this study, 88 randomized controlled trials were analyzed, including a total of 2805 patients and 3073 sockets. The biomaterials/biologics used were self-healing materials, xenografts, allografts, alloplasts, platelet concentrates, and combinations of these biomaterials. Xenografts and allografts, either alone or combined with bioactive agents, were found to be the most effective in preserving horizontal and vertical ridge dimensions. Platelet concentrates were shown to be the best in increasing the percentage of new bone formation. However, a previous network meta-analysis review that included six studies found that freeze-dried bone graft plus membrane was the most likely to be effective in reducing bone height remodeling. Meanwhile, autologous bone marrow was determined to be the most likely effective in terms of width remodeling. Specific objective 2: A total of 12 trials underwent both qualitative and quantitative analysis, which involved evaluating 312 sites. The results indicated that the use of autologous soft tissue grafts resulted in improved horizontal changes compared to resorbable membranes. Furthermore, when comparing crosslinked and non-crosslinked membranes, non-crosslinked membranes were found to be statistically superior, as confirmed by histomorphometric network meta-analysis. This study has no previous reviews to compare its specific objective 2. Specific objective 3: In the network meta-analysis (NMA), 11 studies were analyzed. The included studies had moderate levels of bias. The highest-ranking treatment for vertical buccal height was crosslinked collagen membranes with a SUCRA score of 81.8%. Autogenous soft tissue grafts ranked highest in horizontal width change with a SUCRA score of 89.1%, while the control group had the highest ranking in keratinized mucosa thickness with a SUCRA score of 85.8%. Specific objective 4: The best performers in enhancing KGW were CAF + connective tissue graft (CTG), CAF + platelet concentrate matrix (PCM), and acellular dermal matrix (ADM). In terms of improving the percentage of root coverage in gingival recession, the highest-ranking materials were CAF + collagen matrix (CM) + gingival fibroblasts (GF), CAF + ADM + platelet rich plasma (PRP), and CAF + ADM. These materials outperformed CAF alone. Specific objective 5: Of the 1437 studies identified, 18 relevant studies were included in the analysis. The total number of patients treated was 612, and 848 abutments were inserted. The network meta-analysis (NMA) found that zirconia abutments had a 83.3% probability of being ranked first in terms of plaque index (PI), 87.0% in bleeding on probing (BOP), and 65.0% in probing depth (PD) outcomes. These results indicate that zirconia abutments generally performed better than titanium and alumina abutments. Specific objective 6: 24 studies were included for qualitative analysis and 21 studies for quantitative analysis, published up until 2020. The combined total number of animals in the control and test groups was 162 and 339, respectively. The study duration ranged from 3 to 102 weeks. In the SUCRA rankings, rhBMP-2 was associated with the best performance for bone volume density. rhGDF-5/TCP had the best ranking in bone area (mm2), rhPDGF-BB/Equine in bone height (mm), rhBMP-2 in the percentage of new bone fill, rhBMP-2/ACS in new cementum formation, and rhGDF-5/b- TCP/PLGA in connective tissue attachment and junctional epithelium. Specific objective 7: This study considered 12 clinical studies for qualitative and quantitative analysis. The network meta-analysis found that the combination of rhFGF and hyaluronic acid had the highest ranking in terms of probing pocket depth (PPD) and clinical attachment level (CAL) outcomes. The combination of rhPDGF-BB and β-tricalcium phosphate was ranked highest in terms of percentage of bone filling. Furthermore, all bioactive agents showed better performance compared to control groups without rhGFs. Specific objective 8: In the majority of outcomes, connective tissue graft (CTG) performed the best. When it comes to increasing keratinized mucosa, free gingival graft (FGG) was found to be the best option. Both FGG and crosslinked collagen membranes (XCM) performed better in augmenting keratinized mucosa. As for increasing soft tissue in the buccal aspects, the best results were seen with vascularized connective tissue matrix (VCMX) compared to other matrices. Specific objective 9: 67 studies were eligible for a network meta-analysis (NMA). The study included 1955 patients who underwent 2405 sinus augmentation procedures. The biomaterials used were grouped into: autogenous bone (Auto), xenografts (XG), allografts (AG), alloplasts (AP), bioactive agents (Bio), hyaluronic acid (HA), and combinations of these. A statistically significant inconsistency factor (IF) was found in the entire loop of XG, AP, and Bio+AP. The highest ranked biomaterials for the residual bone height (RBH) of less than 4 mm were XG+AG, XG+AP, and Auto. Similarly, the biomaterials with the highest surface under the cumulative ranking curve (SUCRA) for RBH of 4 mm or more were Auto, Bio+XG, and XG+Auto. Conclusions The rankings of biomaterials can differ depending on the specific clinical outcomes being evaluated. Therefore, it is important to consider all confounding factors and utilize more robust NMA methods to achieve more reliable rankings. The availability of predictive intervals for the majority of research questions enables clinicians to select the most effective biomaterials for future clinical studies, which promotes informed decision-making and reduces costs for patients. However, new evidence continues to emerge through clinical trials and non-randomized studies. As a result, diverse methods such as multidimensional scale ranking, cluster plots for different outcomes, and predictive intervals should be employed when reporting the results of network meta-analyses.