The primary goals of this study were to develop a cell-free in vitro assay for the assessment of nonthermal electromagnetic (EMF) bioeffects and to develop theoretical models in accord with current experimental observations. Based upon the hypothesis that EMF effects operate by modulating Ca2+/CaM binding, an in vitro nitric oxide (NO) synthesis assay was developed to assess the effects of a pulsed radiofrequency (PRF) signal used for treatment of postoperative pain and edema. No effects of PRF on NO synthesis were observed. Effects of PRF on Ca2+/CaM binding were also assessed using a Ca2+-selective electrode, also yielding no EMF Ca2+/CaM binding. However, a PRF effect was observed on the interaction of hemoglobin (Hb) with tetrahydrobiopterin, leading to the development of an in vitro Hb deoxygenation assay, showing a reduction in the rate of Hb deoxygenation for exposures to both PRF and a static magnetic field (SMF). Structural studies using pyranine fluorescence, Gd3+ vibronic sideband luminescence and attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy were conducted in order to ascertain the mechanism of this EMF effect on Hb. Also, the effect of SMF on Hb oxygen saturation (SO2) was assessed under gas-controlled conditions. These studies showed no definitive changes in protein/solvation structure or SO2 under equilibrium conditions, suggesting the need for real-time instrumentation or other means of observing out-of-equilibrium Hb dynamics. Theoretical models were developed for EMF transduction, effects on ion binding, neuronal spike timing, and dynamics of Hb deoxygenation. The EMF sensitivity and simplicity of the Hb deoxygenation assay suggest a new tool to further establish basic biophysical EMF transduction mechanisms. If an EMF-induced increase in the rate of deoxygenation can be demonstrated in vivo, then enhancement of oxygen delivery may be a new therapeutic method by which clinically relevant EMF-mediated enhancement of growth and repair processes can occur.

Over the last two decades, Ostreopsis cf. ovata blooms have been reported with increased frequency, intensity and areal distribution in the Mediterranean Sea. The epiphytic/benthic dinoflagellate proliferates under low hydrodinamism, and produces toxins (palytoxin-like compounds) causing human health problems and massive mortalities of benthic invertebrate communities. While several studies have assessed some effects of abiotic factors like hydrodynamics, water temperature and inorganic nutrients on O. cf. ovata bloom dynamics, biotic factors have been barely addressed. In an effort to provide insights about competition with other microalgal species and microalgal-bacterial interactions in affecting O. cf. ovata growth, physiology and ultimately bloom dynamics, this thesis investigated: (i) the inorganic nutrients uptake and organic phosphorus utilization by O. cf. ovata; (ii) the phylogenetic structure of bacterial assemblages co-occurring with O. cf. ovata bloom in situ; (iii) the microbial (bacteria and viruses) dynamics and toxin production along the O. cf. ovata growth in batch cultures. Data collected show the high efficiency of O. cf. ovata in both inorganic and organic phosphate acquisition, an aspect that could confer advantages towards competing species co-occurring in the microphytobenthos. Recurrent functional associations between O. cf. ovata and Alphaproteobacteria have been evidenced both in situ and in batch cultures, and indicate the latter as a reliable tool for future investigations on O. cf. ovata-bacteria interactions. The ability of some O. cf. ovata-associated Roseobacters (i.e. Ruegeria, Jannaschia, Dinoroseobacter, Roseovarius) to switch from mutualistic to antagonistic relationships depending on algal physiological status suggests an important role of dinoflagellate-bacterial interactions in bloom dynamics. Furthermore, while the vast majority of studies on harmful algal blooms to date have focused on macronutrients’ dynamics, data in this thesis pave the way for further investigations on the role of allochthonous vitamins in O. cf. ovata bloom development and maintenance.

L'ingegneria tissutale è un campo multidisciplinare in rapida crescita che si avvale delle scienze fisiche, ingegneristiche e della vita per sviluppare ed ottenere cellule funzionali, tessuti ed organi per riparare, sostituire o migliorare le funzioni biologiche perse a causa di anomalie congenite, traumi, malattie o invecchiamento. Nell’ambito della ricostruzione della cartilagine articolare sono stati fatti notevoli passi in avanti ma la soluzione per il ripristino completo del tessuto sembra ancora essere lontana. Nella prima parte di questo lavoro è stata valutata la capacità di uno scaffold a base di gelatina di indirizzare le ADSCs verso un differenziamento in senso condrogenico. Successivamente, con lo scopo di migliorare il grado differenziamento e diminuire i costi associati all’utilizzo di fattori differenziativi, l’attenzione è stata posta sullo sviluppo di un biomateriale a base di alginato e ioni cobalto in modo da mimare e sfruttare le caratteristiche fisiche della cartilagine piuttosto che il suo intorno chimico. In ultimo, è stato sviluppato un sistema low cost per la produzione di chip microfluidici sfruttabili per la realizzazione di sistemi micrometrici per incapsulazione cellulare. / Tissue engineering is an interdisciplinary and multidisciplinary field that aims at the developmentof biological substitutes that restore, mantain, or improve tissue function. Concerning the articular cartilage many improvments were made, but the complete tissue restoration approach still lacking. In the first part of this work, it was evaluated the ability of a gelatin scaffold to promote the condrogenic differentiation of ADSCs. Successively, in order to obtain a low cost sistem, a based alginate/Cobalt scaffold was designed with the aim to take advantage of the physical features of the cartilage tissue. Finally, it was developted a cost effective method to produce microfluidic chips with the aim to obtain micro-systems for cell encapsulation.

Global climate changes, driven by increasing temperature and pCO2, negatively affect marine ecosystem and mainly calcifying organisms. The reproduction of the zooxanthellate coral, Balanophyllia europaea, varied significantly along a latitudinal gradient of temperature and solar radiation. The warmer populations showed a lower reproductive efficiency due to the inhibition of the zooxanthellae photosynthesis, leading less available energy for reproduction. On the contrary, the B. europaea population, naturally living along a pCO2 gradient, did not show differences in oocytes development, production and morphology, probably by reallocating additional energy due to increasing of photosynthetic efficiency of zooxanthellae under pCO2 conditions. Increasing pCO2 negatively influenced spermaries production and development in L. pruvoti, causing a delayed in fertilization process, probably due to the lack of zooxanthellae involving in a lack of additional energy available for coral. The effects of high temperature, pCO2 and their interaction were tested on two symbiotic tropical corals, Fungia granulosa (solitary) and Pocillopora verrucosa (colonial). F. granulosa showed negative effects only in interaction treatment, while P. verrucosa showed decreased photosynthetic efficiency, increased bleaching tissue and mortality in all treatments. The more sensitivity of P. verrucosa could be explained by lower tissue thickness, making it more exposed and vulnerable to external environmental conditions. To investigate the role of intra-skeletal organic matrix in coral biomineralization, four Mediterranean species with different trophic strategy and growth form were selected. B. europaea organic matrix presented stronger control and higher independence from the crystallization environment than the other corals. Shell features of clam Chamelea gallina varied along a latitudinal gradient of temperature and solar radiation. Shells from the warmest and the most irradiated population were ~30% lighter due to thinner and more porous shells. No variation was observed in shell polymorphism at the nanoscale level, indicating no effects of environmental parameters on its composition and crystallography.

info:eu-repo/semantics/nonPublished

Several studies have reported beneficial effects of autophagy in preventing chondrocyte death, OA-like changes in gene expression and cartilage degeneration. Many microRNAs have been identified as key modulators of autophagy pathway. So far, to our knowledge no relationship has been revealed between nutraceuticals and microRNA network in OA. First aim of this thesis is to evaluate molecular mechanisms of action of hydroxytyrosol (HT) a promising compound already tested for protective efficacy in OA chondrocytes. HT increases markers of autophagy and protects chondrocytes from DNA damage and cell death induced by oxidative stress. The protective effect requires SIRT-1, which accumulated in the nucleus following HT treatment. Second aim consists in identifying a microRNA implicated in HT-mediated protective response to oxidative stress and examining the effects after modulation of miR levels by approach of transient transfection. After in silico analysis we identify miR-9 as a speculative candidate able to target SIRT-1 and confirm this hypothesis by means of luciferase gene-reporter assay. Moreover miR-9 mediates cell death induced by H2O2 and the protective effect of HT, as observed in human primary chondrocytes and C/28-I2 cell line. Third aim is to investigate the potential role of miR-155, found to be one of the most highly upregulated microRNAs in human OA knee cartilage, in autophagic pathway. Autophagy flux induced by rapamycin and 2-DG was significantly increased by miR-155 LNA, and significantly decreased after miR-155 mimic transfection in T/C28a2 cells and in human primary chondrocytes. These effects of miR-155 on autophagy were related to suppression of gene and protein expression of key autophagy. The identification of a single microRNA, involved simultaneously in several disease-related pathways, discloses a potent therapeutic target. Indeed the unveiling of bioactive compounds, exerting a beneficial effect through induction of epigenetic changes, may open a new topic of research not yet well explored.

In photosynthetic organisms the redox-dependent modification of thiols belonging to enzymes of the photosynthetic cycle, plays a prominent role in the regulation of metabolism and signalling. An important regulatory mechanism is represented by light that acts through a ferredoxin-thioredoxin system. The system permits the reduction/oxidation of disulfide bridges of the target enzymes. Moreover, cysteines residues can undergo other modifications such as glutathionylation and nitrosylation. Proteomic studies have allowed the identification of new putative targets of redox modifications, including Calvin Benson cycle enzymes. The aim of the studies was to investigate whether phosphoglycerate kinase, triose phosphate isomerase and glyceraldehyde-3-phosphate dehydrogenase from Chlamydomonas reinhardtii undergo redox modifications. All enzymes and their mutants (i.e. cysteines variants) were purified and the treatments with alkylating and oxidative agents have permitted to confirm the presence of reactive cysteine(s). The sensitivity of recombinant proteins to redox modifications and the cysteine(s) involved were analyzed by biochemical approcheas. The structural features were analyzed, and the crystallography structure of CrTPI and CrGPA were solved. The three enzymes result all redox regulated although with different biochemical features. The CrPGK contains two cysteines sensitive to redox treatments, although the inhibitor effects of these modifications are different, indeed glutathionylation slightly affected the enzymatic activity compare to nitrosylation. Moreover, the crystallographic structure of CrTPI was determined at a resolution of 1.1.Å, showing a homodimeric conformation containing the typical α/β- barrel fold. No evidence for CrTPI Trx-dependent regulation was obtained but was found to undergo glutathionylation and nitrosylation with a moderate down-regulation on activity. Furthermore, the CrGAPA shows an extreme sensitivity to oxidant molecules and the crystallographic structure of CrGAPA was determined at a resolution of 1.8.Å, confirming the tetrameric fold of the enzyme. the results suggest that redox modifications could constitute a mechanism for the regulation of the Calvin-Benson cycle under oxidative stress conditions.

We performed an innovative systematic meta-analysis of gene expression profiles of whole normal human brain and heart to provide a quantitative transcriptome reference map of it, i.e. a typical reference value of expression for all the known, mapped and uncharacterized (unmapped) transcripts. For this reason, we used the software named TRAM (Transcriptome Mapper), which is able to generate transcriptome maps based on gene expression data from multiple sources. We also analyzed differential gene expression by comparing brain with human foetal brain, with a pool of non-brain tissues and with the three brain sub-region: cerebellum, cerebral cortex and hippocampus, the main regions severely affected with cognitive impairment, as seen in the case of trisomy 21. Data were downloaded from microarray databases, processed and analyzed using TRAM software and validated in vitro by assaying gene expression through several magnitude orders by "Real Time" reverse transcription polymerase chain reaction (RT-PCR). The excellent agreement between in silico and experimental data suggested that our transcriptome maps may be a useful quantitative reference benchmark for gene expression studies related to the human brain and heart. We also generated an integrated quantitative transcriptome map by systematic meta-analysis from all available gene expression profile datasets related to AMKL in paediatric age. The incidence of Acute Megakaryoblastic Leukemia (AMKL) is 500-fold higher in children with Down Syndrome (DS) compared with non-DS children. We present an integrated original model of the DS AMLK transcriptome, providing the identification of genes relevant for its pathophysiology which can potentially be new clinical markers. Finally, computational and molecular analysis of a highly restricted region of chromosome 21, which represents a strong candidate for typical DS features and is considered as intergenic, was performed. Northern Blot analysis and computational biology results show that HR-DSCR contain active loci bidirectionally transcribed.

Chronic obstructive pulmonary disease (COPD) is a multifactorial disease characterized by airflow obstruction that is usually progressive and associated with an abnormal inflammatory response of the lungs to noxious particles and gases. Cigarette smoke (CS) is the main risk factor, but only a small proportion of smokers (15-20%) develop symptomatic disease, this suggests that there are individual susceptibility factors involved in disease onset and progression. Considering the impact of environmental and genetic risk factors in COPD, this dissertation sought to uncover genetic susceptibility biomarkers in a population affected by COPD and explore tissue metabolic alterations induced by chronic CS exposure in a mouse model. A case-control study was carried on in a COPD population, aiming to investigate whether polymorphisms of microsomal epoxide hydrolase (EPHX1) gene, and related phenotypes, had any bearing on individual susceptibility to COPD onset and severity. DNA of COPD patients and controls was genotyped for functional polymorphisms of EPHX1 gene (exon 3 Tyr113His, exon 4 His139Arg), and haplotype analysis was performed using PCR-RFLP and PCR-RT techniques. The statistical analysis did not show any significant result about the potential relationship between analyzed SNPs, related phenotypes, and COPD risk and severity. Stable isotope-resolved metabolomics approach was used to study glycolytic pathway alterations induced by chronic CS exposure in lung and liver tissue of an emphysema mouse model. C57BL/6J mice, after CS exposure, were injected via IP injection with glucose tracer containing carbon stable isotope - 13C6-glucose – then, tissues were collected and the incorporation of 13C into metabolites was monitored by mass spectrometry and nuclear magnetic resonance spettroscopy. Lung tissue analyses did not reveal any significant alteration in lung tissue glycolysis of mice exposed to CS. On the other hand, CS may contribute to dysregulated glycolysis, PPP, glycogen synthesis and utilization, in emphysema mouse model liver tissue.

This study presents the development of a Model of Intermediate Complexity for Ecosystem assessments (MICE) for testing management scenarios for the stock of European hake (Merluccius merluccius) and Norway lobster (Nephrops norvegicus) inhabiting the Central Adriatic Sea. These two species represent the target species, as well as the predators, of this study. They are mainly fished by Italian and Croatian bottom trawl fishery, accounting for both the highest landings and commercial value among demersal species in the area. The specific circulation of the Adriatic Sea is responsible for strong nutrient recycling processes within this area, resulting in a rich fishing ground extensively exploited by Italian and Croatian fishing vessels, make it an interesting case study for the development of an Ecosystem Approach to Fisheries (EAF). Here, an EAF was developed using the MICE approach, since it is well adapted to test specific management questions, as required by this study. The MICE was developed in a step-wise manner. Firstly, single-species stock assessments for both the target species were pursued, allowing the evaluation of the state of these resources and permitting the production of the input values to be included within the MICE. Secondly, the diet of the target species were examined and prey items portioned in four groups; consumption of each prey group by each predator was modelled using a Holling Type II functional response. Finally, predator and prey dynamics were linked within the MICE model, using a modified biomass dynamic model to project them into the future and testing different management strategies. The best management scenario resulted to be the protection of adults, which produced the most beneficial results for both predators and prey groups.