Zinco, invecchiamento e sistema immunitario: effetti sull'apoptosi e sulla proliferazione dei linfociti

Immunosenescence is characterized by a complex remodelling of the immune system, mainly driven by lifelong antigenic burden. Cells of the immune system are constantly exposed to a variety of stressors capable of inducing apoptosis, including antigens and reactive oxygen species continuously produced during immune response and metabolic pathways. The overall homeostasis of the immune system is based on the balance between antigenic load, oxidative stress, and apoptotic processes on one side, and the regenerative potential and renewal of the immune system on the other. Zinc is an essential trace element playing a central role on the immune function, being involved in many cellular processes, such as cell death and proliferation, as cofactor of enzymes, nuclear factors and hormones. In this context, the age associated changes in the immune system may be in part due to zinc deficiency, often observed in aged subjects and able to induce impairment of several immune functions. Thus, the aim of this work was to investigate the role of zinc in two essential events for immunity during aging, i.e. apoptosis and cell proliferation. Spontaneous and oxidative stress-induced apoptosis were evaluated by flow cytometry in presence of a physiological concentration of zinc in vitro on peripheral blood mononuclear cells (PBMCs) obtained from healthy subjects of different age: a group of young subjects, a group of old subjects and a group of nonagenarians. In addition, cell cycle phases were analyzed by flow cytometry in PBMCs, obtained from the subjects of the same groups in presence of different concentration of zinc. We also analyzed the influence of zinc in these processes in relation to p53 codon 72 polymorphism, known to affect apoptosis and cell cycle in age-dependent manner. Zinc significantly reduces spontaneous apoptosis in all age-groups; while it significantly increases oxidative stress-induced late apoptosis/necrosis in old and nonagenarians subjects. Some factors involved in the apoptotic pathway were studied and a zinc effect on mitochondrial membrane depolarization, cytochrome C release, caspase-3 activation, PARP cleavage and Bcl-2 expression was found. In conclusion, zinc inhibits spontaneous apoptosis in PBMCs contrasting the harmful effects due to the cellular culture conditions. On the other hand, zinc is able to increase toxicity and induce cell death in PBMCs from aged subjects when cells are exposed to stressing agents that compromise antioxidant cellular systems. Concerning the relationship between the susceptibility to apoptosis and p53 codon 72 genotype, zinc seems to affect apoptosis only in PBMCs from Pro- people suggesting a role of this ion in strengthening the mechanism responsible of the higher propensity of Pro- towards apoptosis. Regarding cell cycle, high doses of zinc could have a role in the progression of cells from G1 to S phase and from S to G2/M phase. These effect seems depend on the age of the donor but seems to be unrelated to p53 codon 72 genotype. In order to investigate the effect of an in vivo zinc supplementation on apoptosis and cell cycle, PBMCs from a group of aged subjects were studied before and after six weeks of oral zinc supplementation. Zinc supplementation reduces spontaneous apoptosis and it strongly reduces oxidative stress-induced apoptosis. On the contrary, no effect of zinc was observed on cell cycle. Therefore, it’s clear that in vitro and in vivo zinc supplementation have different effects on apoptosis and cell cycle in PBMCs from aged subjects. Further experiments and clinical trials are necessary to clarify the real effect of an in vivo zinc supplementation because this preliminary data could encourage the of this element in all that disease with oxidative stress pathogenesis. Moreover, the expression of metallothioneins (MTs), proteins well known for their zinc-binding ability and involved in many cellular processes, i.e. apoptosis, metal ions detoxification, oxidative stress, differentiation, was evaluated in total lymphocytes, in CD4+ and in CD8+ T lymphocytes from young and old healthy subjects in presence of different concentration of zinc in vitro. Literature data reported that during ageing the levels of these proteins increase and concomitantly they lose the ability to release zinc. This fact induce a down-regulation of many biological functions related to zinc, such as metabolism, gene expression and signal transduction. Therefore, these proteins may turn from protective in young-adult age to harmful agents for the immune function in ageing following the concept that several genes/proteins that increase fitness early in life may have negative effects later in life: named “Antagonistic Pleyotropy Theory of Ageing”. Data obtained in this work indicate an higher and faster expression of MTs with lower doses of zinc in total lymphocytes, in CD4+ and in CD8+ T lymphocytes from old subjects supporting the antagonistic pleiotropic role of these proteins.

Physiology and Biotechnology of the Hydrogen Production with the Green Microalga Chlamydomonas reinhardtii

The hydrogen production in the green microalga Chlamydomonas reinhardtii was evaluated by means of a detailed physiological and biotechnological study. First, a wide screening of the hydrogen productivity was done on 22 strains of C. reinhardtii, most of which mutated at the level of the D1 protein. The screening revealed for the first time that mutations upon the D1 protein may result on an increased hydrogen production. Indeed, productions ranged between 0 and more than 500 mL hydrogen per liter of culture (Torzillo, Scoma et al., 2007a), the highest producer (L159I-N230Y) being up to 5 times more performant than the strain cc124 widely adopted in literature (Torzillo, Scoma, et al., 2007b). Improved productivities by D1 protein mutants were generally a result of high photosynthetic capabilities counteracted by high respiration rates. Optimization of culture conditions were addressed according to the results of the physiological study of selected strains. In a first step, the photobioreactor (PBR) was provided with a multiple-impeller stirring system designed, developed and tested by us, using the strain cc124. It was found that the impeller system was effectively able to induce regular and turbulent mixing, which led to improved photosynthetic yields by means of light/dark cycles. Moreover, improved mixing regime sustained higher respiration rates, compared to what obtained with the commonly used stir bar mixing system. As far as the results of the initial screening phase are considered, both these factors are relevant to the hydrogen production. Indeed, very high energy conversion efficiencies (light to hydrogen) were obtained with the impeller device, prooving that our PBR was a good tool to both improve and study photosynthetic processes (Giannelli, Scoma et al., 2009). In the second part of the optimization, an accurate analysis of all the positive features of the high performance strain L159I-N230Y pointed out, respect to the WT, it has: (1) a larger chlorophyll optical cross-section; (2) a higher electron transfer rate by PSII; (3) a higher respiration rate; (4) a higher efficiency of utilization of the hydrogenase; (5) a higher starch synthesis capability; (6) a higher per cell D1 protein amount; (7) a higher zeaxanthin synthesis capability (Torzillo, Scoma et al., 2009). These information were gathered with those obtained with the impeller mixing device to find out the best culture conditions to optimize productivity with strain L159I-N230Y. The main aim was to sustain as long as possible the direct PSII contribution, which leads to hydrogen production without net CO2 release. Finally, an outstanding maximum rate of 11.1 ± 1.0 mL/L/h was reached and maintained for 21.8 ± 7.7 hours, when the effective photochemical efficiency of PSII (ΔF/F'm) underwent a last drop to zero. If expressed in terms of chl (24.0 ± 2.2 µmoles/mg chl/h), these rates of production are 4 times higher than what reported in literature to date (Scoma et al., 2010a submitted). DCMU addition experiments confirmed the key role played by PSII in sustaining such rates. On the other hand, experiments carried out in similar conditions with the control strain cc124 showed an improved final productivity, but no constant PSII direct contribution. These results showed that, aside from fermentation processes, if proper conditions are supplied to selected strains, hydrogen production can be substantially enhanced by means of biophotolysis. A last study on the physiology of the process was carried out with the mutant IL. Although able to express and very efficiently utilize the hydrogenase enzyme, this strain was unable to produce hydrogen when sulfur deprived. However, in a specific set of experiments this goal was finally reached, pointing out that other than (1) a state 1-2 transition of the photosynthetic apparatus, (2) starch storage and (3) anaerobiosis establishment, a timely transition to the hydrogen production is also needed in sulfur deprivation to induce the process before energy reserves are driven towards other processes necessary for the survival of the cell. This information turned out to be crucial when moving outdoor for the hydrogen production in a tubular horizontal 50-liter PBR under sunlight radiation. First attempts with laboratory grown cultures showed that no hydrogen production under sulfur starvation can be induced if a previous adaptation of the culture is not pursued outdoor. Indeed, in these conditions the hydrogen production under direct sunlight radiation with C. reinhardtii was finally achieved for the first time in literature (Scoma et al., 2010b submitted). Experiments were also made to optimize productivity in outdoor conditions, with respect to the light dilution within the culture layers. Finally, a brief study of the anaerobic metabolism of C. reinhardtii during hydrogen oxidation has been carried out. This study represents a good integration to the understanding of the complex interplay of pathways that operate concomitantly in this microalga.

Intensificazione di processi biologici per la Bioremediation aerobica di suoli contaminati

Enzyveba, a partially characterized complex consortium of not-adapted microorganisms developed through prolonged stabilization of organic wastes, was found to markedly intensify the aerobic remediation of aged PAH- and PCB-contaminated soil by acting as a source of exogenous specialized microorganisms and nutrients. Thus, Enzyveba was tested in the bioremediation of Diesel (G1) and HiQ Diesel (G2) contaminated soils under aerobic slurry-phase conditions by means of a chemical, microbiological, ecotoxicological integrated analytical procedure. The addition of Enzyveba resulted in a higher availability of cultivable specialized bacteria and fungi but this resulted in a slight intensification of soil remediation, probably because of the high content of nutrients and specialized microorganisms of the soil. In many cases, the biotreatability of soils impacted by diesel fuel is limited by their poor content of autochthonous pollutant-degrading microorganisms. Thus, bioaugmentation with stable and reproducible cultures with the required broad substrate specificity might be the solution for a successful remediation. Two microbial consortia, ENZ-G1 and ENZ-G2, were enriched from Enzyveba on G1 and G2. Both consortia consist of a similar composition of bacterial and fungal species. They exhibited a comparable and significant biodegradation capability by removing about 90% of 1 g/l of diesel fuel under liquid culture conditions. Given their remarkable biodegradation potential, richness of quite diverse microbes, stability and resistance after cryopreservation at -20 °C for several months, both consortia appear very interesting candidates for bioaugmentation on site. The mycoflora of a soil historically contaminated by high concentration of PCBs was characterised before, at the beginning and at the end of the biotreatment mentioned above. Several mitosporic fungi isolated from soil grew in presence of a mixture of three PCBs congeners when also glucose was provided. This is the first study in which 5 strains of mitosporic species able to biodegrade PCB are reported in the literature.

Mesenchymal stromal cell: new applications for regenerative medicine

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

Toward a 3D in vitro model based on decellularized thymus to maintain adult thymic ephitelial cells functionality

During my PhD,I have been develop an innovative technique to reproduce in vitro the 3D thymic microenvironment, to be used for growth and differentiation of thymocytes, and possible transplantation replacement in conditions of depressed thymic immune regulation. The work has been developed in the laboratory of Tissue Engineering at the University Hospital in Basel, Switzerland, under the tutorship of Prof.Ivan Martin. Since a number of studies have suggested that the 3D structure of the thymic microenvironment might play a key role in regulating the survival and functional competence of thymocytes, I’ve focused my effort on the isolation and purification of the extracellular matrix of the mouse thymus. Specifically, based on the assumption that TEC can favour the differentiation of pre-T lymphocytes, I’ve developed a specific decellularization protocol to obtain the intact, DNA-free extracellular matrix of the adult mouse thymus. Two different protocols satisfied the main characteristics of a decellularized matrix, according to qualitative and quantitative assays. In particular, the quantity of DNA was less than 10% in absolute value, no positive staining for cells was found and the 3D structure and composition of the ECM were maintained. In addition, I was able to prove that the decellularized matrixes were not cytotoxic for the cells themselves, and were able to increase expression of MHC II antigens compared to control cells grown in standard conditions. I was able to prove that TECs grow and proliferate up to ten days on top the decellularized matrix. After a complete characterization of the culture system, these innovative natural scaffolds could be used to improve the standard culture conditions of TEC, to study in vitro the action of different factors on their differentiation genes, and to test the ability of TECs to induce in vitro maturation of seeded T lymphocytes.

Microglial involvement in brain physiopathology: in vitro studies using rat primary cultures

Microglial involvement in neurological disorders is well-established, being microglial activation not only associated with neurotoxic consequences, but also with neuroprotective effects. The studies presented here, based on microglia rat primary cell cultures and mainly on microglial conditioned medium (MCM), show insights into the mechanism of Superoxide dismutase 1 (SOD1) and Apolipoprotein E (ApoE) secretion by microglia as well as their neuroprotective effect towards primary cerebellar granule neurons (CGNs) exposed to the dopaminergic toxin 6-hydroxydopamine (6-OHDA). SOD1 and ApoE are released respectively through non-classical lysosomal or the classical ER/Golgi-mediated secretion pathway. Microglial conditioned medium, in which SOD1 and ApoE accumulated, protected CGNs from degeneration and these effects were replicated when exogenous SOD1 or ApoE was added to a non-conditioned medium. SOD1 neuroprotective action was mediated by increased cell calcium from an external source. ApoE release is negatively affected by microglia activation, both with lipopolysaccharide (LPS) and Benzoylbenzoyl-ATP (Bz-ATP) but is stimulated by neuronal-conditioned medium as well as in microglia-neurons co-culture conditions. This neuronal-stimulated microglial ApoE release is differently regulated by activation states (i.e. LPS vs ATP) and by 6-hydroxydopamine-induced neurodegeneration. In co-culture conditions, microglial ApoE release is essential for neuroprotection, since microglial ApoE silencing through siRNA abrogated protection of cerebellar granule neurons against 6-OHDA toxicity. Therefore, these molecules could represent a target for manipulation aimed at promoting neuroprotection in brain diseases. Considering a pathological context, and the microglial ability to adopt a neuroprotective or neurotoxic profile, we characterize the microglial M1/M2 phenotype in transgenic rats (McGill-R-Thy1-APP) which reproduce extensively the Alzheimer’s-like amyloid pathology. Here, for the first time, cortical, hippocampal and cerebellar microglia of wild type and transgenic adult rats were compared, at both early and advanced stages of the pathology. In view of possible therapeutic translations, these findings are relevant to test microglial neuroprotection, in animal models of neurodegenerative diseases.

Mesenchymal Stromal Cells (MSCs) and induced Plutipotent Stem Cells (iPSCs) in Domestic Animals: Characterization and Differentiation Potential

Derivation of stem cell lines from domesticated animals has been of great interest as it benefits translational medicine, clinical applications to improve human and animal health and biotechnology. The main types of stem cells studied are Embryonic Stem Cells (ESCs), induced Pluripotent Stem Cells (iPSCs) and Mesenchymal Stem/Stromal Cells (MSCs). This thesis had two main aims: (I) The isolation of bovine MSCs from amniotic fluid (AF) at different trimesters of pregnancy and their characterization to study pluripotency markers expression. Stemness markers were studied also in MSCs isolated from equine AF, Wharton’s jelly (WJ) and umbilical cord blood (UCB) as continuation of the characterization of these cells previously performed by our research group; (II) The establishment and characterization of iPSCs lines in two attractive large animal models for biomedical and biotechnology research such as the bovine and the swine, and the differentiation into the myogenic lineage of porcine iPSCs. It was observed that foetal tissues in domestic animals such as the bovine and the horse represent a source of MSCs able to differentiate into the mesodermal lineage but they do not proliferate indefinitely and they lack the expression of many pluripotency markers, making them an interesting source of cells for regenerative medicine, but not the best candidate to elucidate pluripotency networks. The protocol used to induce pluripotency in bovine fibroblasts did not work, as well as the chemical induction of pluripotency in porcine fibroblasts, while the reprogramming protocol used for porcine iPSCs was successful and the line generated was amenable to being differentiated into the myogenic lineage, demonstrating that they could be addressed into a desired lineage by genetic modification and appropriated culture conditions. Only a few cell types have been differentiated from domestic animal iPSCs to date, so the development of a reliable directed-differentiation protocol represents a very important result.