HIF1α regulates Mitochondrial biogenesis and Cellular senescence induced by Gamma Radiation

Cellular response to γ-rays is mediated by ATM-p53 axis. When p53 is phosphorylated, it can transactivate several genes to induce permanent cell cycle arrest (senescence) or apoptosis. Epithelial and mesenchymal cells are more resistant to radiation-induced apoptosis and respond mainly by activating senescence. Hence, tumor cells in a senescent state might remain as “dormant” malignant in fact through disruption of p53 function, cells may overcome growth arrest. Oncocytic features were acquired in the recurring neoplasia after radiation therapy in patient with colonrectal cancer. Oncocytic tumors are characterized by aberrant biogenesis and are mainly non-aggressive neoplasms. Their low proliferation degree can be explained by chronic destabilization of HIF1α, which presides to adaptation to hypoxia and also plays a pivotal role in hypoxia-related radio-resistance. The aim of the present thesis was to verify whether mitochondrial biogenesis can be induced following radiation treatment, in relation of HIF1α status and whether is predictive of a senescence response. In this study was demonstrate that mitochondrial biogenesis parameters like mitochondrial DNA copy number could be used for the prediction of hypoxic status of tissue after radiation treatment. γ-rays induce an increase of mitochondrial mass and function, in response to a genotoxic stress that pushes cells into senescence. Mitochondrial biogenesis is only indirectly regulated by p53, whose activation triggers a MDM2-mediated HIF1α degradation, leading to the release of PGC-1β inhibition by HIF1α. On the other hand, this protein blunts the mitochondrial response to γ-rays as well as the induction of p21-mediated cell senescence, indicating prevalence of the hypoxic over the genotoxic response. Finally in vivo, post-radiotherapy mtDNA copy number increase well correlates with lack of HIF1α increase in the tissue, concluding this may be a useful molecular tool to infer the trigger of a hypoxic response during radiotherapy, which may lead to failure of activation of senescence.

Evaluation of the transcriptomic response of an Alzheimer's disease murine model induced at different ages: searching for predictors

Alzheimer’s disease (AD) is a chronic, progressive neurodegenerative disease, characterized by the impairment of mnesic and cognitive functions, that represents the most frequent type of dementia in older people worldwide. Aging is the most important risk factor for the sporadic form of the pathology and it is associated to the progressive impairment of the proteostasis network. The endoplasmic reticulum (ER), the main cellular actor involved in proteostasis, appears significantly compromised in AD due to the accumulation of β-amyloid (Aβ) protein and phosphorylated-tau protein. Increasing proteins misfolding activates a specific cellular response known as Unfolded Protein response (UPR) which orchestrates the recovery of ER function. The aim of the present study was to investigate the role of UPR and aging process in a murine model of AD induced by intracerebroventricular (i.c.v.) injection of Aβ1-42 oligomers at 3 or 18 months. The oligomers injection in aged animals caused the increased of memory impairment, oxidative stress, and the depletion of glutathione reserve. Furthermore, the RNA-sequencing analysis was performed and the bioinformatic analysis showed the enrichment of several pathways involved in neurodegeneration and protein regulations. The following analysis highlighted the significant dysregulation of the three branches of the UPR, the protein kinase RNA-like ER kinase (PERK), inositol-requiring protein 1α (IRE1α) and activating transcription factor 6 (ATF-6). In turn, ER stress affected the PI3K/Akt/Gsk3β and MAPK/ERK pathways, highlighting Mapkapk5 as a potential marker of the neurodegenerative process, which regulation could lead to the definition of new pharmacological and neuroprotective strategies to counteract AD.

Peripheral arterial disease and diabetes: effects on endothelial progenitor cell differentiation and nitric oxide metabolism

In the recent years it is emerged that peripheral arterial disease (PAD) has become a growing health problem in Western countries. This is a progressive manifestation of atherothrombotic vascular disease, which results into the narrowing of the blood vessels of the lower limbs and, as final consequence, in critical leg ischemia. PAD often occurs along with other cardiovascular risk factors, including diabetes mellitus (DM), low-grade inflammation, hypertension, and lipid disorders. Patients with DM have an increased risk of developing PAD, and that risk increases with the duration of DM. Moreover, there is a growing population of patients identified with insulin resistance (IR), impaired glucose tolerance, and obesity, a pathological condition known as “metabolic syndrome”, which presents increased cardiovascular risk. Atherosclerosis is the earliest symptom of PAD and is a dynamic and progressive disease arising from the combination of endothelial dysfunction and inflammation. Endothelial dysfunction is a broad term that implies diminished production or availability of nitric oxide (NO) and/or an imbalance in the relative contribution of endothelium-derived relaxing factors. The secretion of these agents is considerably reduced in association with the major risks of atherosclerosis, especially hyperglycaemia and diabetes, and a reduced vascular repair has been observed in response to wound healing and to ischemia. Neovascularization does not only rely on the proliferation of local endothelial cells, but also involves bone marrow-derived stem cells, referred to as endothelial progenitor cells (EPCs), since they exhibit endothelial surface markers and properties. They can promote postnatal vasculogenesis by homing to, differentiating into an endothelial phenotype, proliferating and incorporating into new vessels. Consequently, EPCs are critical to endothelium maintenance and repair and their dysfunction contributes to vascular disease. The aim of this study has been the characterization of EPCs from healthy peripheral blood, in terms of proliferation, differentiation and function. Given the importance of NO in neovascularization and homing process, it has been investigated the expression of NO synthase (NOS) isoforms, eNOS, nNOS and iNOS, and the effects of their inhibition on EPC function. Moreover, it has been examined the expression of NADPH oxidase (Nox) isoforms which are the principal source of ROS in the cell. In fact, a number of evidences showed the correlation between ROS and NO metabolism, since oxidative stress causes NOS inactivation via enzyme uncoupling. In particular, it has been studied the expression of Nox2 and Nox4, constitutively expressed in endothelium, and Nox1. The second part of this research was focused on the study of EPCs under pathological conditions. Firstly, EPCs isolated from healthy subject were cultured in a hyperglycaemic medium, in order to evaluate the effects of high glucose concentration on EPCs. Secondly, EPCs were isolated from the peripheral blood of patients affected with PAD, both diabetic or not, and it was assessed their capacity to proliferate, differentiate, and to participate to neovasculogenesis. Furthermore, it was investigated the expression of NOS and Nox in these cells. Mononuclear cells isolated from peripheral blood of healthy patients, if cultured under differentiating conditions, differentiate into EPCs. These cells are not able to form capillary-like structures ex novo, but participate to vasculogenesis by incorporation into the new vessels formed by mature endothelial cells, such as HUVECs. With respect to NOS expression, these cells have high levels of iNOS, the inducible isoform of NOS, 3-4 fold higher than in HUVECs. While the endothelial isoform, eNOS, is poorly expressed in EPCs. The higher iNOS expression could be a form of compensation of lower eNOS levels. Under hyperglycaemic conditions, both iNOS and eNOS expression are enhanced compared to control EPCs, as resulted from experimental studies in animal models. In patients affected with PAD, the EPCs may act in different ways. Non-diabetic patients and diabetic patients with a higher vascular damage, evidenced by a higher number of circulating endothelial cells (CECs), show a reduced proliferation and ability to participate to vasculogenesis. On the other hand, diabetic patients with lower CEC number have proliferative and vasculogenic capacity more similar to healthy EPCs. eNOS levels in both patient types are equivalent to those of control, while iNOS expression is enhanced. Interestingly, nNOS is not detected in diabetic patients, analogously to other cell types in diabetics, which show a reduced or no nNOS expression. Concerning Nox expression, EPCs present higher levels of both Nox1 and Nox2, in comparison with HUVECs, while Nox4 is poorly expressed, probably because of uncompleted differentiation into an endothelial phenotype. Nox1 is more expressed in PAD patients, diabetic or not, than in controls, suggesting an increased ROS production. Nox2, instead, is lower in patients than in controls. Being Nox2 involved in cellular response to VEGF, its reduced expression can be referable to impaired vasculogenic potential of PAD patients.

Percezione della direzione del proprio movimento: dalla registrazione dell'attività corticale al modello computazionale.

The main aim of this thesis is strongly interdisciplinary: it involves and presumes a knowledge on Neurophysiology, to understand the mechanisms that undergo the studied phenomena, a knowledge and experience on Electronics, necessary during the hardware experimental set-up to acquire neuronal data, on Informatics and programming to write the code necessary to control the behaviours of the subjects during experiments and the visual presentation of stimuli. At last, neuronal and statistical models should be well known to help in interpreting data. The project started with an accurate bibliographic research: until now the mechanism of perception of heading (or direction of motion) are still poorly known. The main interest is to understand how the integration of visual information relative to our motion with eye position information happens. To investigate the cortical response to visual stimuli in motion and the integration with eye position, we decided to study an animal model, using Optic Flow expansion and contraction as visual stimuli. In the first chapter of the thesis, the basic aims of the research project are presented, together with the reasons why it’s interesting and important to study perception of motion. Moreover, this chapter describes the methods my research group thought to be more adequate to contribute to scientific community and underlines my personal contribute to the project. The second chapter presents an overview on useful knowledge to follow the main part of the thesis: it starts with a brief introduction on central nervous system, on cortical functions, then it presents more deeply associations areas, which are the main target of our study. Furthermore, it tries to explain why studies on animal models are necessary to understand mechanism at a cellular level, that could not be addressed on any other way. In the second part of the chapter, basics on electrophysiology and cellular communication are presented, together with traditional neuronal data analysis methods. The third chapter is intended to be a helpful resource for future works in the laboratory: it presents the hardware used for experimental sessions, how to control animal behaviour during the experiments by means of C routines and a software, and how to present visual stimuli on a screen. The forth chapter is the main core of the research project and the thesis. In the methods, experimental paradigms, visual stimuli and data analysis are presented. In the results, cellular response of area PEc to visual stimuli in motion combined with different eye positions are shown. In brief, this study led to the identification of different cellular behaviour in relation to focus of expansion (the direction of motion given by the optic flow pattern) and eye position. The originality and importance of the results are pointed out in the conclusions: this is the first study aimed to investigate perception of motion in this particular cortical area. In the last paragraph, a neuronal network model is presented: the aim is simulating cellular pre-saccadic and post-saccadic response of neuron in area PEc, during eye movement tasks. The same data presented in chapter four, are further analysed in chapter fifth. The analysis started from the observation of the neuronal responses during 1s time period in which the visual stimulation was the same. It was clear that cells activities showed oscillations in time, that had been neglected by the previous analysis based on mean firing frequency. Results distinguished two cellular behaviour by their response characteristics: some neurons showed oscillations that changed depending on eye and optic flow position, while others kept the same oscillations characteristics independent of the stimulus. The last chapter discusses the results of the research project, comments the originality and interdisciplinary of the study and proposes some future developments.

Ribosome-inactivating proteins and their immunotoxins for cancer therapy: insights into the mechanism of cell death

Ribosome-inactivating proteins (RIPs) are a family of plant toxic enzymes that permanently damage ribosomes and possibly other cellular substrates, thus causing cell death involving different and still not completely understood pathways. The high cytotoxic activity showed by many RIPs makes them ideal candidates for the production of immunotoxins (ITs), chimeric proteins designed for the selective elimination of unwanted or malignant cells. Saporin-S6, a type 1 RIP extracted from Saponaria officinalis L. seeds, has been extensively employed to construct anticancer conjugates because of its high enzymatic activity, stability and resistance to conjugation procedures, resulting in the efficient killing of target cells. Here we investigated the anticancer properties of two saporin-based ITs, anti-CD20 RTX/S6 and anti-CD22 OM124/S6, designed for the experimental treatment of B-cell NHLs. Both ITs showed high cytotoxicity towards CD20-positive B-cells, and their antitumor efficacy was enhanced synergistically by a combined treatment with proteasome inhibitors or fludarabine. Furthermore, the two ITs showed differencies in potency and ability to activate effector caspases, and a different behavior in the presence of the ROS scavenger catalase. Taken together, these results suggest that the different carriers employed to target saporin might influence saporin intracellular routing and saporin-induced cell death mechanisms. We also investigated the early cellular response to stenodactylin, a recently discovered highly toxic type 2 RIP representing an interesting candidate for the design and production of a new IT for the experimental treatment of cancer.

Role of Reactive Oxygen Species in signalling and oxidative stress

Results reported in this Thesis contribute to the comprehension of the complicated world of “redox biology”. ROS regulate signalling pathways both in physiological responses and in pathogenesis and progression of diseases. In cancer cells, the increase in ROS generation from metabolic abnormalities and oncogenic signalling may trigger a redox adaptation response, leading to an up-regulation of antioxidant capacity in order to maintain the ROS level below the toxic threshold. Thus, cancer cells would be more dependent on the antioxidant system and more vulnerable to further oxidative stress induced by exogenous ROS-generating agents or compounds that inhibit the antioxidant system. Results here reported indicate that the development of new drugs targeting specific Nox isoforms, responsible for intracellular ROS generation, or AQP isoforms, involved in the transport of extracellular H2O2 toward intracellular targets, might be an interesting novel anti-leukaemia strategy. Furthermore, also the use of CSD peptide, which simulate the VEGFR-2 segregation into caveolae in the inactive form, might be a strategy to stop the cellular response to VEGF signalling. As above stated, in the understanding of the redox biology, it is also important to identify and distinguish the molecular effectors that maintain normal biological and physiological responses, such as agents that stimulate our adaptation systems and elevate our endogenous antioxidant defences or other protective systems. Data here reported indicate that the nutraceutical compound sulforaphane and the Klotho protein are able to stimulate the HO-1 and Prx-1 expression, as well as the GSH levels, confirming their antioxidant and protective role. Finally, results here reported demonstrated that Stevia extracts are involved in insulin regulated glucose metabolism, suggesting that the use of these compounds goes beyond their sweetening power and may also offer therapeutic benefits hence improving the quality of life.

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

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

Analysis of the memory B cell response against glycoconjugate vaccines

The development of vaccines directed against polysaccharide capsules of S. pneumoniae, H. influenzae and N. meningitidis have been of great importance in preventing potentially fatal infections. Bacterial capsular polysaccharides are T-cell-independent antigens that induce specific antibody response characterized by IgM immunoglobulins, with a very low IgG class switched response and lack of capability of inducing a booster response. The inability of pure polysaccharides to induce sustained immune responses has required the development of vaccines containing polysaccharides conjugated to a carrier protein, with the aim to generate T cell help. It is clear that the immunogenicity of glycoconjugate vaccines can vary depending on different factors, e.g. chemical nature of the linked polysaccharide, carrier protein, age of the target population, adjuvant used. The present study analyzes the memory B cell (MBC) response to the polysaccharide and to the carrier protein following vaccination with a glycoconjugate vaccine for the prevention of Group B streptococcus (GBS) infection. Not much is known about the role of adjuvants in the development of immunological memory raised against GBS polysaccharides, as well as about the influence of having a pre-existing immunity against the carrier protein on the B cell response raised against the polysaccharide component of the vaccine. We demonstrate in the mouse model that adjuvants can increase the antibody and memory B cell response to the carrier protein and to the conjugated polysaccharide. We also demonstrate that a pre-existing immunity to the carrier protein favors the development of the antibody and memory B cell response to subsequent vaccinations with a glycoconjugate, even in absence of adjuvants. These data provide a useful insight for a better understanding of the mechanism of action of this class of vaccines and for designing the best vaccine that could result in a productive and long lasting memory response.

From Folke Medicine to Medicine Chemistry: Study, Using in Vitro and Cellular Assays, of Receptors Mechanism Involved in the Activities of Natural Compounds

My Doctorate Research has been focused on the evaluation of the pharmacological activity of a natural extract of chestnut wood (ENC) towards the cardiovascular and gastrointestinal system and on the identification of the active compounds. The ENC has been shown to contain more than 10% (w/w) of phenolic compounds, of which tannins as Vescalgin and Castalgin are the more representative. ENC cardiovascular effects have been investigated in guinea pig cardiac preparations; furthermore its activity has been evalueted in guinea pig aorta strips. ENC induced transient negative chronotropic effect in isolated spontaneously beating right atria and simultaneously positive inotropic effect in left atria driven at 1 Hz. Cardiac cholinergic receptors are not involved in the negative chronotropic effect and positive inotropic effects are not related to adrenergic receptors. In vascular smooth muscle, natural extract of chestnut did not significantly change the contraction induced by potassium (80 mM) or that induced by noradrenaline (1μM). In guinea pig ileum, ENC reduced the maximum response to carbachol in a concentrationdependent manner and behaved as a reversible non competitive antagonist. In guinea pig ileum, the antispasmodic activity of ENC showed a significant antispasmodic activity against a variety of different spasmogenic agents including histamine, KCl, BaCl2. In guinea pig proximal colon, stomach and jejunum, ENC reduced the maximum response to carbachol in a concentrationdependent manner and behaved as a reversible non competitive antagonist. ENC contracted gallbladder guinea pig in a reversible and concentration-dependent manner. This effect does not involve cholinergic and cholecystokinin receptors and it is reduced by nifedipine. ENC relaxed Oddi sphincter smooth muscle. The cholecystokinetic and Oddi sphincter relaxing activities occurred also in guinea pigs fed a lithogenic diet. The cholecystokinetic occurred also in human gallbladder. The Fractionation of the extract led to the identification of the active fraction.

Phytoplankton Response to Environmental Variables and Organic Pollutants. Laboratory Cultures and Numerical Simulations Experiments

Herbicides are becoming emergent contaminants in Italian surface, coastal and ground waters, due to their intensive use in agriculture. In marine environments herbicides have adverse effects on non-target organisms, as primary producers, resulting in oxygen depletion and decreased primary productivity. Alterations of species composition in algal communities can also occur due to the different sensitivity among the species. In the present thesis the effects of herbicides, widely used in the Northern Adriatic Sea, on different algal species were studied. The main goal of this work was to study the influence of temperature on algal growth in the presence of the triazinic herbicide terbuthylazine (TBA), and the cellular responses adopted to counteract the toxic effects of the pollutant (Chapter 1 and 2). The development of simulation models to be applied in environmental management are needed to organize and track information in a way that would not be possible otherwise and simulate an ecological prospective. The data collected from laboratory experiments were used to simulate algal responses to the TBA exposure at increasing temperature conditions (Chapter 3). Part of the thesis was conducted in foreign countries. The work presented in Chapter 4 was focused on the effect of high light on growth, toxicity and mixotrophy of the ichtyotoxic species Prymnesium parvum. In addition, a mesocosm experiment was conducted in order to study the synergic effect of the pollutant emamectin benzoate with other anthropogenic stressors, such as oil pollution and induced phytoplankton blooms (Chapter 5).

In vitro study of the osteocytes response to hypoxia and their regulation of bone homeostasis

Bone remodelling is a fundamental mechanism for removing and replacing bone during adaptation of the skeleton to mechanical loads. Skeletal unloading leads to severe hypoxia (1%O2) in the bone microenvironment resulting in imbalanced bone remodelling that favours bone resorption. Hypoxia, in vivo, is a physiological condition for osteocytes, 5% O2 is more likely physiological for osteocytes than 20% O2, as osteocytes are embedded deep inside the mineralized bone matrix. Osteocytes are thought to be the mechanosensors of bone and have been shown to orchestrate bone formation and resorption. Oxygen-deprived osteocytes seem undergo apoptosis and actively stimulate osteoclasts. Hypoxia and oxidative stress increase 150-kDa oxygen-regulated protein (ORP 150) expression in different cell types. It is a novel endoplasmic-reticulum-associated chaperone induced by hypoxia/ischemia. It well known that ORP 150 plays an important role in the cellular adaptation to hypoxia, as anti-apoptotic factor, and seems to be involved in osteocytes differentiations. The aims of the present study are 1) to determine the cellular and molecular response of the osteocytes at two different conditions of oxygen deprivation, 1% and 5% of O2 compared to the atmospheric oxygen concentration at several time points. 2) To clarify the role of hypoxic osteocytes in bone homeostasis through the detection of releasing of soluble factors (RANKL, OPG, PGE2 and Sclerostin). 3) To detect the activation of osteoclast and osteoblast induced by condition media collected from hypoxic and normoxic osteocytes. The data obtained in this study shows that hypoxia compromises the viability of osteocytes and induces apoptosis. Unlike in other cells types, ORP 150 in MLO-Y4 does not seem to be regulated early during hypoxia. The release of soluble factors and the evaluation of osteoclast and osteoblast activation shows that osteocytes, grown under severe oxygen deprivation, play a role in the regulation of both bone resorption and bone formation.

Salinity Effect on Horticultural Crops: Morphological, Physiological, and Biomolecular Elements of Salinity Stress Response

Among abiotic stresses, high salinity stress is the most severe environmental stress. High salinity exerts its negative impact mainly by disrupting the ionic and osmotic equilibrium of the cell. In saline soils, high levels of sodium ions lead to plant growth inhibition and even death. Salt tolerance in plants is a multifarious phenomenon involving a variety of changes at molecular, organelle, cellular, tissue as well as whole plant level. In addition, salt tolerant plants show a range of adaptations not only in morphological or structural features but also in metabolic and physiological processes that enable them to survive under extreme saline environments. The main objectives of my dissertation were understanding the main physiological and biomolecular features of plant responses to salinity in different genotypes of horticultural crops that are belonging to different families Solanaceae (tomato) and Cucurbitaceae (melon) and Brassicaceae (cabbage and radish). Several aspects of crop responses to salinity have been addressed with the final aim of combining elements of functional stress response in plants by using several ways for the assessment of plant stress perception that ranging from destructive measurements (eg. leaf area, relative growth rate, leaf area index, and total plant fresh and dry weight), to physiological determinations (eg. stomatal conductance, leaf gas exchanges, water use efficiency, and leaf water relation), to the determination of metabolite accumulation in plant tissue (eg. Proline and protein) as well as evaluation the role of enzymatic antioxidant capacity assay in scavenging reactive oxygen species that have been generated under salinized condition, and finally assessing the gene induction and up-down regulation upon salinization (eg. SOS pathway).

Molecular basis of Osteoarthritis and aspects of cellular senescence in disease

The aim of this study is to investigate on some molecular mechanisms contributing to the pathogenesis of osteoarthritis (OA) and in particular to the senescence of articular chondrocytes. It is focused on understanding molecular events downstream GSK3β inactivation or dependent on the activity of IKKα, a kinase that does not belong to the phenotype of healthy articular chondrocytes. Moreover, the potential of some nutraceuticals on scavenging ROS thus reducing oxidative stress, DNA damage, and chondrocyte senescence has been evaluated in vitro. The in vitro LiCl-mediated GSK3β inactivation resulted in increased mitochondrial ROS production, that impacted on cellular proliferation, with S-phase transient arrest, increased SA-β gal and PAS staining, cell size and granularity. ROS are also responsible for the of increased expression of two major oxidative lesions, i.e. 1) double strand breaks, tagged by γH2AX, that associates with activation of GADD45β and p21, and 2) 8-oxo-dG adducts, that associate with increased IKKα and MMP-10 expression. The pattern observed in vitro was confirmed on cartilage from OA patients. IKKa dramatically affects the intensity of the DNA damage response induced by oxidative stress (H2O2 exposure) in chondrocytes, as evidenced by silencing strategies. At early time point an higher percentage of γH2AX positive cells and more foci in IKKa-KD cells are observed, but IKKa KD cells proved to almost completely recover after 24 hours respect to their controls. Telomere attrition is also reduced in IKKaKD. Finally MSH6 and MLH1 genes are up-regulated in IKKαKD cells but not in control cells. Hydroxytyrosol and Spermidine have a great ROS scavenging capacity in vitro. Both treatments revert the H2O2 dependent increase of cell death and γH2AX-foci formation and senescence, suggesting the ability of increasing cell homeostasis. These data indicate that nutraceuticals represent a great challenge in OA management, for both therapeutical and preventive purposes.

A preclinical study of spinal cord injury focused on cellular and molecular modifications as potential targets for innovative therapies

Spinal Cord Injury (SCI) is a devastating condition for human and animal health. In SCI particularly, neurons, oligodendrocytes precursor cells, and mature oligodendrocytes are highly vulnerable to the toxic microenvironment after the lesion and susceptible to the elevated levels of noxious stimuli. Thus the regenerative response of the organism in case of SCI is significantly reduced, and only little spontaneous amelioration is observed in lesioned patients during the early phases. This work mainly focuses on studying and characterizing the modification induced by the SCI in a preclinical animal model. We investigated the ECM composition in the spinal cord segments surrounding the primary lesion site at a gene expression level. We found Timp1 and CD44 as a crucial hub in the secondary cascade of SCI in both spinal cord segments surrounding the lesion site. Interestingly, a temporal and anatomical difference in gene expression, indicating a complex regulation of ECM genes after SCI that could be used as a tool for regenerative medicine. We also investigated the modification in synaptic plasticity-related gene expression in spinal and supraspinal areas involved in motor control. We confirmed the anatomical and temporal difference in gene expression in spinal cord tissue. This analysis suggests that a molecular mapping of the lesion-induced modification could be a useful tool for regenerative medicine. In the last part, we evaluated the efficacy of an implantable biopolymer loaded with an anti-inflammatory drug and a pro-myelinating agent on the acute phase of SCI in our preclinical model. We found a consistent reduction of the inflammatory state in the spinal lesion site and the cord's surrounding segments. Moreover, we found increased preservation of the spinal cord tissue with a related upregulation of neuronal and oligodendroglial markers after lesion. Our treatment showed effective ameliorating functional outcome and reducing the lesion extension in the chronic phase.

Evaluation of the pathogenetic role of cellular subsets of the Follicular Lymphoma Microenvironment

Follicular lymphoma (FL) is a B cell neoplasm, composed of follicle center cells, that accounts for about 20% of all lymphomas, with the highest incidence reported in the USA and western Europe. FL has been considered a virtually incurable disease, with a high response rate alternated with frequent post-therapy relapses or progression towards more aggressive lymphomas. Due to the extreme variability in outcome, many efforts were made to predict prognosis, the need for therapy, and the likelihood of evolution. Even if clinical scores turned out to be robust and easy to use in clinical practice for patient risk stratification, marked heterogeneity in outcome remains within each group and further insights into the biology of FL are needed. The genome-wide approach underscored the pivotal role of the FL microenvironment in the evolution of the disease. In 2004, a landmark study by Dave et al. first described the microenvironment impact on tumor biology. By gene expression profiling they identified two different immune response signatures, involving T-cells and macrophages which seemed to independently predict FL outcome, but their exact is not completely understood and different studies led to variable results. Subsequently, many workgroups identified in amount and distribution pattern of these different cell subsets features which can impact prognosis, this leading to hypothesizing the use of these parameters as surrogate markers of the molecular signature. We aimed to assess the possible contributions of micro-environmental components to FL transformation or progression, its relevance as a prognostic/predictive tool, and its potential role as an innovative therapeutic target. We used immunohistochemical techniques, focusing specifically on macrophages and T-cells subsets, and then found correlations between the presence, proportions, and distribution of these reactive cells and the clinical outcomes leading to the future development of a reliable tool for upfront risk stratification of patients affected by FL.