Functional and molecular characterization of ethylene responsive factor genes involved in grape ripening

Grape berry is considered a non climacteric fruit, but there are some evidences that ethylene plays a role in the control of berry ripening. This PhD thesis aimed to give insights in the role of ethylene and ethylene-related genes in the regulation of grape berry ripening. During this study a small increase in ethylene concentration one week before véraison has been measured in Vitis vinifera L. ‘Pinot Noir’ grapes confirming previous findings in ‘Cabernet Sauvignon’. In addition, ethylene-related genes have been identified in the grapevine genome sequence. Similarly to other species, biosynthesis and ethylene receptor genes are present in grapevine as multi-gene families and their expression appeared tissue or developmental specific. All the other elements of the ethylene signal transduction cascade were also identified in the grape genome. Among them, there were ethylene response factors (ERF) which modulate the transcription of many effector genes in response to ethylene. In this study seven grapevine ERFs have been characterized and they showed tissue and berry development specific expression profiles. Two sequences, VvERF045 and VvERF063, seemed likely involved in berry ripening control due to their expression profiles and their sequence annotation. VvERF045 was induced before véraison and was specific of the ripe berry, by sequence similarity it was likely a transcription activator. VvERF063 displayed high sequence similarity to repressors of transcription and its expression, very high in green berries, was lowest at véraison and during ripening. To functionally characterize VvERF045 and VvERF063, a stable transformation strategy was chosen. Both sequences were cloned in vectors for over-expression and silencing and transferred in grape by Agrobacterium-mediated or biolistic-mediated gene transfer. In vitro, transgenic VvERF045 over-expressing plants displayed an epinastic phenotype whose extent was correlated to the transgene expression level. Four pathogen stress response genes were significantly induced in the transgenic plants, suggesting a putative function of VvERF045 in biotic stress defense during berry ripening. Further molecular analysis on the transgenic plants will help in identifying the actual VvERF045 target genes and together with the phenotypic characterization of the adult transgenic plants, will allow to extensively define the role of VvERF045 in berry ripening.

The regulation of Satellite Cells during skeletal muscle regeneration and neuromuscular disease.

Skeletal muscle possesses the remarkable capacity to complete a rapid and extensive regeneration, even following severe damage. The regenerative ability of skeletal muscle relies on Satellite Cells (SCs), a population of muscle specific adult stem cells. However, during aging or under several pathological conditions, the ability of skeletal muscle to fully regenerated is compromised. Here, a morphological and molecular study on SCs from patients affected by ALS is described. Moreover, the role of the cell cycle regulator P16Ink4a during skeletal muscle regeneration and aging has been investigated.

Cellular and Molecular Biomarkers for Risk Assessment of Colorectal cancer

The detection of Colorectal Cancer (CRC), at early stages, is one of the proven strategies resulting in a higher cure rate. In recent years, several studies have appeared identifying potential cancer markers in serum, plasma and stool in an attempt to improve actual screening procedures. Thus, the aim of the study was (1) Evaluate MN frequency, (2) Evaluate plasma ultrafiltrate capacity to induce MN formation, (3) Evaluate SEPT9 and NOTCH3 promoter methylation profile in peripheral blood lymphocytes from subjects resulted positive to fecal occult blood test and examined by colonoscopy. MN frequency was significantly higher in subjects with histological diagnosis of CRC and adenoma than control (p ≤ 0.001 and p ≤ 0.01, respectively). About, CF-MN analysis, a statistically significant difference was observed between CRC and control (p ≤ 0.05). On the other hand, SEPT9 and NOTCH3 promoter methylation status was significantly lower in CRC subjects than controls; additionally, NOTCH3 promoter methylation status was significantly lower in CRC subjects than adenoma subjects (p ≤ 0.01). The results obtained allow conclude that MN frequency varies according CRC pathologic status and, together with other variables, is a valid biomarker for adenoma and CRC risk. Additionally, the plasma of patients affected with CRC not only serve as a biomarker for oxidative stress but also as biomarker of genetic damage correlated with the carcinogenic process that verifies in colon-rectum. SEPT9 and NOTCH3 promoter methylation status, at peripheral blood level, varies according hystopathological changes observed in colon-rectum, suggesting that promoter methylation profile of these genes could be a reliable biomarker for CRC risk.

Cellular and molecular effects of cross reacting material 197 (CRM197) on HT-29 colon cancer cell line

Cross Reacting Material 197(CRM197) is a Diphteria toxin non toxic mutant that had shown anti-tumor activity in mice and humans. CRM197 is utilized as a specific inhibitor of heparin-binding epidermal growth factor (HB-EGF), that competes for the epidermal growth factor receptor (EGFR), overexpressed in colorectal cancer and implicated in its progression. We evaluated the effects of CRM197 on HT-29 human colon cancer cell line behaviour and, for CRM197 recognized ability to inhibit HB-EGF, its possible effects on EGFR activation. In particular, while HT-29 does not show any reduction of viability after CRM197 treatment, or changes in cell cycle distribution, in EGFR localization or activation, they show a change in gene expression profile analyzed by microarray. This is the first study where the CRM197 treatment on HT-29 show the alteration of a specific and selected number of genes.

Understanding The Physiological, Biochemical, and Molecular Mechanisms of Salinity Tolerance in Strawberry Cultivars and in HvTPK1-Overexpressed Barley

The research was carried out to investigate of main elements of salt stress response in two strawberry cultivars, Elsanta and Elsinore. Plants were grown under 0, 10, 20 and 40 mM NaCl for 80 days. Salinity dramatically affected growth in both cultivars, although Elsinore appeared to be more impaired than Elsanta. Moreover a significant reduction of leaf photosynthesis, evaporation, and stomatal conductance was recorded 24 hrs after the stress was applied in both cultivars, whereas physiological functions were differentially restored after acclimation. However, cv. Elsanta had more efficient leaf gas exchange and water status than cv. Elsinore. In general, Fruit yield reduced upon salinization, wheares fruit quality concerning fruit taste, aroma, appearance, total soluble solids and titratable acidity, did not change but rather was enhanced under moderate salinity. On the other hand fruit quality was impaired at severe salt stress. Fruit antioxidant content and antioxidant capacity were enhanced significantly by increasing salt concentration in both cultivars. The oxidative effects of the stress were defined by the measures of some enzymatic activities and lipid peroxidation. Consistently, an increase in superoxide dismutase (SOD), catalase (CAT), peroxide dismutase (POD) enzymes and higher content of proline and soluble proteins were observed in cv. Elsinore than in cv. Elsanta. The increase coincided with a decrease in lipid peroxidation. The research confirmed that although strawberry cultivars were sensitive to salinity, difference between cultivars exist; The experiment revealed that cv. Elsanta could stand severe salt stress, which was lethal to cv. Elsinore. The parameters measured in the previous experiment were proposed as early screening tools for the salt stress response in nine strawberry genotypes. The results showed that, wheares Elsanta and Elsinore cultivars had a lower dry weight reduction at 40 mM NaCl among cultivars, Naiad, Kamila, and Camarosa were the least salt-sensitive cultivars among the screened.

Effects of Global Warming on Berry Composition of cv. Sangiovese: Biochemical and Molecular Aspects and Agronomical Adaptation Approaches

Wine grape must deal with serious problems due to the unfavorable climatic conditions resulted from global warming. High temperatures result in oxidative damages to grape vines. The excessive elevated temperatures are critical for grapevine productivity and survival and contribute to degradation of grape and wine quality and yield. Elevated temperature can negatively affect anthocyanin accumulation in red grape. Particularly, cv. Sangiovese was identified to be very sensitive to such condition. The quantitative real-time PCR analysis showed that flavonoid biosynthetic genes were slightly repressed by high temperature. Also, the heat stress repressed the expression of the transcription factor “VvMYBA1” that activates the expression of UFGT. Moreover, high temperatures had repressing effects on the activity of the flavonoids biosynthetic enzymes “PAL” and “UFGT”.Anthocyanin accumulation in berry skin is due to the balance between its synthesis and oxidation. In grape cv. Sangiovese, the gene transcription and activity of peroxidases enzyme was elevated by heat stress as a defensive mechanism of ROS-scavenging. Among many isoforms of peroxidases genes, one gene (POD 1) was induced in Sangiovese under thermal stress condition. This gene was isolated and evaluated via the technique of genes transformation from grape to Petunia. Reduction in anthocyanins concentration and higher enzymatic activity of peroxidase was observed in POD 1 transformed Petunia after heat shock compared to untrasformed control. Moreover, in wine producing regions, it is inevitable for the grape growers to adopt some adaptive strategies to alleviate grape damages to abiotic stresses. Therefore, in this thesis, the technique of post veraison trimming was done to improve the coupling of phenolic and sugar ripening in Vitis vinifera L. cultivar Sangiovese. Trimming after veraison showed to be executable to slow down the rate of sugar accumulation in grape (to decrease the alcohol potential in wines) without evolution of the main berry flavonoids compounds.

Phenotypic and molecular characterization of ethylene biosynthesis in apples

Ethylene plays an important role in apple fruit development. Its biosynthesis is catalyzed by two enzymes ACS and ACO. The first is considered to catalyzes the rate-limiting step of ethylene production and in apple two different alleles (MdACS1-1 and MdACS1-2) of this gene have been identified. The presence in the promoter region of MdACS1-2 allele of a SINE insertion is considered to be responsible for a low transcription level and a pronounced reduction in ethylene production in apple cultivar homozygous for this allele. However, the specific expression of each MdACS1 allele has never been reported as well as any in vivo analysis of its 5’-flanking region. With the present study we addressed these issues by developing a set of qPCR allele specific primers for MdACS1 and by a functional characterization of the MdACS1 promoters by transient expression analysis. qPCR analysis on different apple tissues and stages of development demonstrated that MdACS1-2 allele is never express and that MdACS1-1 allele is ripening-related and expresses predominantly but not exclusively in apple fruit. To test MdACS1 promoter in fruit the only protocol available in literature for transient transformation of apple fruit was evaluated and optimized. Twenty chimeric promoter::reporter constructs were generated and analyzed by Agrobacterium-transient transformation. The in vivo analysis allowed to identify an enhancer-like region of 261 bp in MdACS1 promoter and a region of 57 bp in MdACS1-2 responsible, also if not alone, in the inactivation of the MdACS1-2 allele. Through the assessment of ethylene production in a segregating progeny derived from the cross between Fuji and Mondial Gala (homozygous for MdACS1-2 allele) we demonstrated that at least two other genes may be involved in apple ethylene production. An hypothesis that could explain the difference between Fuji and Mondial Gala have been proposed.

Studies of pure rotational spectra of isolated molecules and molecular adducts using pulsed jet Fourier transform microwave (PJ-FTM) spectroscopy

This thesis focuses on studying molecular structure and internal dynamics by using pulsed jet Fourier transform microwave (PJ-FTMW) spectroscopy combined with theoretical calculations. Several kinds of interesting chemical problems are investigated by analyzing the MW spectra of the corresponding molecular systems. First, the general aspects of rotational spectroscopy are summarized, and then the basic theory on molecular rotation and experimental method are described briefly. ab initio and density function theory (DFT) calculations that used in this thesis to assist the assignment of rotational spectrum are also included. From chapter 3 to chapter 8, several molecular systems concerning different kind of general chemical problems are presented. In chapter 3, the conformation and internal motions of dimethyl sulfate are reported. The internal rotations of the two methyl groups split each rotational transition into several components line, allowing for the determination of accurate values of the V3 barrier height to internal rotation and of the orientation of the methyl groups with respect to the principal axis system. In chapter 4 and 5, the results concerning two kinds of carboxylic acid bi-molecules, formed via two strong hydrogen bonds, are presented. This kind of adduct is interesting also because a double proton transfer can easily take place, connecting either two equivalent or two non-equivalent molecular conformations. Chapter 6 concerns a medium strong hydrogen bonded molecular complex of alcohol with ether. The dimer of ethanol-dimethylether was chosen as the model system for this purpose. Chapter 7 focuses on weak halogen…H hydrogen bond interaction. The nature of O-H…F and C-H…Cl interaction has been discussed through analyzing the rotational spectra of CH3CHClF/H2O. In chapter 8, two molecular complexes concerning the halogen bond interaction are presented.

Epidemiology and population genetics of Podosphaera fusca and Golovinomyces orontii, causal agents of cucurbit powdery mildew

In 2010, 2011 and 2012 growing seasons, the occurrence of the ascomycetes Podosphaera fusca and Golovinomyces orontii, causal agents of powdery mildew disease, was monitored on cultivated cucurbits located in Bologna and Mantua provinces to determine the epidemiology of the species. To identify the pathogens, both morphological and molecular identifications were performed on infected leaf samples and a Multiplex-PCR was performed to identify the mating type genes of P. fusca isolates. The investigations indicated a temporal succession of the two species with the earlier infections caused by G. orontii, that seems to be the predominant species till the middle of July when it progressively disappears and P. fusca becomes the main species infecting cucurbits till the end of October. The temporal variation is likely due to the different overwintering strategies of the two species instead of climatic conditions. Only chasmothecia of P. fusca were recorded and mating type alleles ratio tended to be 1:1. Considering that only chasmothecia of P. fusca were found, molecular-genetic analysis were carried out to find some evidence of recombination within this species by MLST and AFLP methods. Surprisingly, no variations were observed within isolates for the 8 MLST markers used. According to this result, AFLP analysis showed a high similarity within isolates, with SM similarity coefficient ranging between 0.91-1.00 and also, sequencing of 12 polymorphic bands revealed identity to some gene involved in mutation and selection. The results suggest that populations of P. fusca are likely to be a clonal population with some differences among isolates probably due to agricultural practices such as fungicides treatments and cultivated hosts. Therefore, asexual reproduction, producing a lot of fungal biomass that can be easily transported by wind, is the most common and useful way to the spread and colonization of the pathogen.

Circulating Fibrocytes, their role in renal fibrosis and molecular pathways involved. Possible biomarkers of fibrogenesis in chronic kidney disease

Circulating Fibrocytes (CFs) are bone marrow-derived mesenchymal progenitor cells that express a similar pattern of surface markers related to leukocytes, hematopoietic progenitor cells and fibroblasts. CFs precursor display an ability to differentiate into fibroblasts and Myofibroblasts, as well as adipocytes. Fibrocytes have been shown to contribute to tissue fibrosis in the end-stage renal disease (ESRD), as well as in other fibrotic diseases, leading to fibrogenic process in other organs including lung, cardiac, gut and liver. This evidence has been confirmed by several experimental proofs in mice models of kidney injury. In the present study, we developed a protocol for the study of CFs, by using peripheral blood monocytes cells (PBMCs) samples collected from healthy human volunteers. Thanks to a flow cytometry method, in vitro culture assays and the gene expression assays, we are able to study and characterize this CFs population. Moreover, results confirmed that these approaches are reliable and reproducible for the investigation of the circulating fibrocytes population in whole blood samples. Our final aim is to confirm the presence of a correlation between the renal fibrosis progression, and the different circulating fibrocyte levels in Chronic Kidney Disease (CKD) patients. Thanks to a protocol study presented and accepted by the Ethic Committee we are continuing the study of CFs induction in a cohort of sixty patients affected by CKD, divided in three distinct groups for different glomerular filtration rate (GFR) levels, plus a control group of thirty healthy subjects. Ongoing experiments will determine whether circulating fibrocytes represent novel biomarkers for the study of CKD progression, in the early and late phases of this disease.

Contribution of Non-Covalent Interactions and Electronic Effects on the Conformational Landscape and Tautomeric Equilibria of Molecules and Molecular Complexes: Structural and Dynamical Data from Rotational Spectroscopy

This thesis concerns the study of complex conformational surfaces and tautomeric equilibria of molecules and molecular complexes by quantum chemical methods and rotational spectroscopy techniques. In particular, the focus of this research is on the effects of substitution and noncovalent interactions in determining the energies and geometries of different conformers, tautomers or molecular complexes. The Free-Jet Absorption Millimeter Wave spectroscopy and the Pulsed-Jet Fourier Transform Microwave spectroscopy have been applied to perform these studies and the obtained results showcase the suitability of these techniques for the study of conformational surfaces and intermolecular interactions. The series of investigations of selected medium-size molecules and complexes have shown how different instrumental setups can be used to obtain a variety of results on molecular properties. The systems studied, include molecules of biological interest such as anethole and molecules of astrophysical interest such as N-methylaminoethanol. Moreover halogenation effects have been investigated on halogen substituted tautomeric systems (5-chlorohydroxypyridine and 6-chlorohydroxypyridine), where it has shown that the position of the inserted halogen atom affects the prototropic equilibrium. As for fluorination effects, interesting results have been achieved investigating some small complexes where a molecule of water is used as a probe to reveal the changes on the electrostatic potential of different fluorinated compounds: 2-fluoropyridine, 3-fluoropyridine and penta-fluoropyridine. While in the case of the molecular complex between water and 2-fluoropyridine and 3-fluoropyridine the geometry of the complex with one water molecule is analogous to that of pyridine with the water molecule linked to the pyridine nitrogen, the case of pentafluoropyridine reveals the effect of perfluorination and the water oxygen points towards the positive center of the pyridine ring. Additional molecular adducts with a molecule of water have been analyzed (benzylamine-water and acrylic acid-water) in order to reveal the stabilizing driving forces that characterize these complexes.