Studies of Inorganic Layer and Framework Structures Using Time-, Temperature- and Pressure-Resolved Powder Diffraction Techniques

This thesis is concerned with in-situ time-, temperature- and pressure-resolved synchrotron X-ray powder diffraction investigations of a variety of inorganic compounds with twodimensional layer structures and three-dimensional framework structures. In particular, phase stability, reaction kinetics, thermal expansion and compressibility at non-ambient conditions has been studied for 1) Phosphates with composition MIV(HPO4)2·nH2O (MIV = Ti, Zr); 2) Pyrophosphates and pyrovanadates with composition MIVX2O7 (MIV = Ti, Zr and X = P, V); 3) Molybdates with composition ZrMo2O8. The results are compiled in seven published papers and two manuscripts. Reaction kinetics for the hydrothermal synthesis of α-Ti(HPO4)2·H2O and intercalation of alkane diamines in α-Zr(HPO4)2·H2O was studied using time-resolved experiments. In the high-temperature transformation of γ-Ti(PO4)(H2PO4)·2H2O to TiP2O7 three intermediate phases, γ'-Ti(PO4)(H2PO4)·(2-x)H2O, β-Ti(PO4)(H2PO4) and Ti(PO4)(H2P2O7)0.5 were found to crystallise at 323, 373 and 748 K, respectively. A new tetragonal three-dimensional phosphate phase called τ-Zr(HPO4)2 was prepared, and subsequently its structure was determined and refined using the Rietveld method. In the high-temperature transformation from τ-Zr(HPO4)2 to cubic α-ZrP2O7 two new orthorhombic intermediate phases were found. The first intermediate phase, ρ-Zr(HPO4)2, forms at 598 K, and the second phase, β-ZrP2O7, at 688 K. Their respective structures were solved using direct methods and refined using the Rietveld method. In-situ high-pressure studies of τ-Zr(HPO4)2 revealed two new phases, tetragonal ν-Zr(HPO4)2 and orthorhombic ω-Zr(HPO4)2 that crystallise at 1.1 and 8.2 GPa. The structure of ν-Zr(HPO4)2 was solved and refined using the Rietveld method. The high-pressure properties of the pyrophosphates ZrP2O7 and TiP2O7, and the pyrovanadate ZrV2O7 were studied up to 40 GPa. Both pyrophosphates display smooth compression up to the highest pressures, while ZrV2O7 has a phase transformation at 1.38 GPa from cubic to pseudo-tetragonal β-ZrV2O7 and becomes X-ray amorphous at pressures above 4 GPa. In-situ high-pressure studies of trigonal α-ZrMo2O8 revealed the existence of two new phases, monoclinic δ-ZrMo2O8 and triclinic ε-ZrMo2O8 that crystallises at 1.1 and 2.5 GPa, respectively. The structure of δ-ZrMo2O8 was solved by direct methods and refined using the Rietveld method.

Variance Reduction in Analytical Chemistry : New Numerical Methods in Chemometrics and Molecular Simulation

This thesis is based on five papers addressing variance reduction in different ways. The papers have in common that they all present new numerical methods. Paper I investigates quantitative structure-retention relationships from an image processing perspective, using an artificial neural network to preprocess three-dimensional structural descriptions of the studied steroid molecules. Paper II presents a new method for computing free energies. Free energy is the quantity that determines chemical equilibria and partition coefficients. The proposed method may be used for estimating, e.g., chromatographic retention without performing experiments. Two papers (III and IV) deal with correcting deviations from bilinearity by so-called peak alignment. Bilinearity is a theoretical assumption about the distribution of instrumental data that is often violated by measured data. Deviations from bilinearity lead to increased variance, both in the data and in inferences from the data, unless invariance to the deviations is built into the model, e.g., by the use of the method proposed in paper III and extended in paper IV. Paper V addresses a generic problem in classification; namely, how to measure the goodness of different data representations, so that the best classifier may be constructed. Variance reduction is one of the pillars on which analytical chemistry rests. This thesis considers two aspects on variance reduction: before and after experiments are performed. Before experimenting, theoretical predictions of experimental outcomes may be used to direct which experiments to perform, and how to perform them (papers I and II). After experiments are performed, the variance of inferences from the measured data are affected by the method of data analysis (papers III-V).

Genetic characterization and molecular identification of the bloodmeal sources of the potential bluetongue vector Culicoides obsoletus in the Canary Islands, Spain

[EN] Background: Culicoides (Diptera: Ceratopogonidae) biting midges are vectors for a diversity of pathogens including bluetongue virus (BTV) that generate important economic losses. BTV has expanded its range in recent decades, probably due to the expansion of its main vector and the presence of other autochthonous competent vectors. Although the Canary Islands are still free of bluetongue disease (BTD), Spain and Europe have had to face up to a spread of bluetongue with disastrous consequences. Therefore, it is essential to identify the distribution of biting midges and understand their feeding patterns in areas susceptible to BTD. To that end, we captured biting midges on two farms in the Canary Islands (i) to identify the midge species in question and characterize their COI barcoding region and (ii) to ascertain the source of their bloodmeals using molecular tools.Methods: Biting midges were captured using CDC traps baited with a 4-W blacklight (UV) bulb on Gran Canaria and on Tenerife. Biting midges were quantified and identified according to their wing patterns. A 688 bp segment of the mitochondrial COI gene of 20 biting midges (11 from Gran Canaria and 9 from Tenerife) were PCR amplified using the primers LCO1490 and HCO2198. Moreover, after selected all available females showing any rest of blood in their abdomen, a nested-PCR approach was used to amplify a fragment of the COI gene from vertebrate DNA contained in bloodmeals. The origin of bloodmeals was identified by comparison with the nucleotide-nucleotide basic alignment search tool (BLAST). Results: The morphological identification of 491 female biting midges revealed the presence of a single morphospecies belonging to the Obsoletus group. When sequencing the barcoding region of the 20 females used to check genetic variability, we identified two haplotypes differing in a single base. Comparison analysis using the nucleotide-nucleotide basic alignment search tool (BLAST) showed that both haplotypes belong to Culicoides obsoletus, a potential BTV vector. As well, using molecular tools we identified the feeding sources of 136 biting midges and were able to confirm that C. obsoletus females feed on goats and sheep on both islands.Conclusions: These results confirm that the feeding pattern of C. obsoletus is a potentially important factor in BTV transmission to susceptible hosts in case of introduction into the archipelago. Consequently, in the Canary Islands it is essential to maintain vigilance of Culicoides-transmitted viruses such as BTV and the novel Schmallenberg virus.

Clinicopathological and molecular characterization of gastroesophageal junction (GEJ) adenocarcinoma before age of 40 years

Gastroesophageal junction (GEJ) adenocarcinoma are uncommon before age of 40 years. While certain clinical, pathological and molecular features of GEJ adenocarcinoma in older patients have been extensively studied, these characteristics in the younger population remain to be determined. In the recent literature, a high sensitivity and specificity for the detection of dysplasia and esophageal adenocarcinoma was demonstrated by using multicolor fluorescence in situ hybridization (FISH) DNA probe set specific for the locus specific regions 9p21 (p16), 20q13.2 and Y chromosome. We evaluated 663 patients with GEJ adenocarcinoma and further divided them into 2 age-groups of and >or= 50 years, rispectively. FISH with selected DNA probe for Y chromosome, locus 9p21 (p16), and locus 20q13.2 was investigated with formalin fixed and parassin embedded tissue from surgical resections of 17 younger and 11 older patients. Signals were counted in > 100 cells with each given histopathological category. The chromosomal aberrations were then compared in the 2 age-groups with the focus on uninvolved squamous and columnar epithelium, intestinal metaplasia (Barrett's mucosa), glandular dysplasia, and adenocarcinoma. Comparisons were performed by the X2 test, Fisher's exact test, Student's t-test and Mann-Whitney U-test as appropriate. Survival was estimated by the Kaplan-Meier method with univariate analysis by the log-rank. Significance was taken at the 5% level. There was no difference in the surgical technique applied in both age groups and most patients underwent Ivor Lewis esophagectomy. Among clinical variables there was a higher incidence of smocking history in older patient group. We identified a progressive loss of Y chromosome from benign squamos epithelium to Barrett's mucosa and glandular dysplasia, and, ultimately, to a near complete loss in adenocarcinoma in both age groups. The young group revealed significantly more losses of 9p21 in both benign and neoplastic cells when compared to the older patients group. In addition, we demonstrated an increase in the percentage of cells showing gain of locus 20q13.2 with progression from benign epithelium through dysplasia to adenocarcinoma with almost the same trend in both the young and the older patients. When compared with the older age-group, younger patients with GEJ adenocarcinoma possess similar known demographics, environmental factors, clinical, and pathologic characteristics. The most commonly detected genetic aberrations of progressive Y chromosomal loss, 9p21 locus loss, and 20q13 gains were similar in the younger and older patients. However the rate of loss of 9p21 is significantly higher in young patients, in both the benign and the neoplastic cells. The loss of 9p21, and possibly, the subsequent inactivation of p16 gene may be one of the molecular mechanisms responsible for the accelerated neoplastic process in young patients.

Salt stress responses in pear and quince: physiological and molecular aspects

The productivity of agricultural crops is seriously limited by salinity. This problem is rapidly increasing, particularly in irrigated lands. Like almost all the fruit tree species, Pyrus communis is generally considered a salt sensitive species, but only little information is available on its behavior under saline conditions. Previous studies, carried out in the Department of Fruit Tree and Woody Plant Science (University of Bologna), focused their attention on pear and quince salt stress responses to understand which rootstock would be the most suitable for pear in order to tolerate a salt stress condition. It has been reported that pear and quince have different ability in the uptake, translocation and accumulation of chloride (Cl-) and sodium (Na+) ions, when plants were irrigated for one season with saline water (5 dS/m). The aim of the present work was to deepen these aspects and investigate salt stress responses in pear and quince. Two different experiments have been performed: a “short-term” trial in a growth chamber and a “long-term” experiment in the open field. In the short-term experiment, three different genotypes usually adopted as pear rootstocks (MC, BA29 and Farold®40) and the pear variety Abbé Fétel own rooted have been compared under salt stress conditions. The trial was performed in a hydroponic culture system, applying a 90 mM NaCl stress to half of the plants, after five weeks of normal growth in Hoagland’s solution. During the three-weeks of salt stress treatment, physiological, mineral and molecular analyses were performed in order to monitor, for each genotype, the development of the salt stress responses in comparison with the corresponding “unstressed” plants. Farold®40 and Abbé Fétel own rooted showed the onset of leaf necrosis, due to salt toxicity, one week before quinces. Moreover, quinces displayed a significant delay in premature senescence of old leaves, while pears emerged for their ability to regenerate new leaves from apparently dead foliage with the salt stress still running. Physiological measurements, such as shoots length, chlorophyll (Chl) content, and photosynthesis, have been carried out and revealed that pears exhibited a significant reduction in water content and a wilting aspect, while for quinces a decrease in Chl content and a growth slowdown were observed. At the end of the trial, all plants were collected and organs separated for dry weight estimation and mineral analyses (Cu, Fe, Mn, Zn Mg, Ca, K, Na and Cl). Mineral contents have been affected by salinity; same macro/micro nutrients were altered in some organs or relocated within the plant. This plant response could have partially contributed to face the salt stress. Leaves and roots have been harvested for molecular analyses at four different times during stress conditions. Molecular analyses consisted of the gene expression study of three main ion transporters, well known in Arabidopsis thaliana as salt-tolerance determinants in the “SOS” pathway: NHX1 (tonoplast Na+/H+ antiporter), SOS1 (plasmalemma Na+/H+ antiporter) and HKT1 (K+ high-affinity and Na+ low-affinity transporter). These studies showed that two quince rootstocks adopted different responsive mechanisms to NaCl stress. BA29 increased its Na+ sequestration activity into leaf vacuoles, while MC enhanced temporarily the same ability, but in roots. Farold®40, instead, exhibited increases in SOS1 and HKT1 expression mainly at leaf level in the attempt to retrieve Na+ from xylem, while Abbé Fétel differently altered the expression of these genes in roots. Finally, each genotype showed a peculiar response to salt stress that was the sum of its ability in Na+ exclusion, osmotic tolerance and tissue tolerance. In the long-term experiment, potted trees of the pear variety Abbé Fétel grafted on different rootstocks (MC, BA29 and Farold®40), or own rooted and also rootstocks only were subjected to a salt stress through saline water irrigation with an electrical conductivity of 5 dS/m for two years. The purposes of this study were to evaluate salinity effects on physiological (shoot length, number of buds, photosynthesis, etc.) and yield parameters of cultivar Abbé Fétel in the different combinations and to determine the salt amount that pear is able to tolerate over the years. With this work, we confirmed the previous hypothesis that pear, despite being classified as a salt-sensitive fruit tree, can be cultivated for two years under saline water irrigation, without showing any salt toxicity symptoms or severe drawbacks on plant development and production. Among different combinations, Abbé Fétel grafted on MC resulted interesting for its peculiar behaviors under salt stress conditions. In the near future, further investigations on physiological and molecular aspects will be necessary to enrich and broaden the knowledge of salt stress responses in pear.

NMR, Metabonomics and Molecular Profiles: Applications to the Quality Assessment of Foodstuffs

Nuclear Magnetic Resonance (NMR) is a branch of spectroscopy that is based on the fact that many atomic nuclei may be oriented by a strong magnetic field and will absorb radiofrequency radiation at characteristic frequencies. The parameters that can be measured on the resulting spectral lines (line positions, intensities, line widths, multiplicities and transients in time-dependent experi-ments) can be interpreted in terms of molecular structure, conformation, molecular motion and other rate processes. In this way, high resolution (HR) NMR allows performing qualitative and quantitative analysis of samples in solution, in order to determine the structure of molecules in solution and not only. In the past, high-field NMR spectroscopy has mainly concerned with the elucidation of chemical structure in solution, but today is emerging as a powerful exploratory tool for probing biochemical and physical processes. It represents a versatile tool for the analysis of foods. In literature many NMR studies have been reported on different type of food such as wine, olive oil, coffee, fruit juices, milk, meat, egg, starch granules, flour, etc using different NMR techniques. Traditionally, univariate analytical methods have been used to ex-plore spectroscopic data. This method is useful to measure or to se-lect a single descriptive variable from the whole spectrum and , at the end, only this variable is analyzed. This univariate methods ap-proach, applied to HR-NMR data, lead to different problems due especially to the complexity of an NMR spectrum. In fact, the lat-ter is composed of different signals belonging to different mole-cules, but it is also true that the same molecules can be represented by different signals, generally strongly correlated. The univariate methods, in this case, takes in account only one or a few variables, causing a loss of information. Thus, when dealing with complex samples like foodstuff, univariate analysis of spectra data results not enough powerful. Spectra need to be considered in their wholeness and, for analysing them, it must be taken in consideration the whole data matrix: chemometric methods are designed to treat such multivariate data. Multivariate data analysis is used for a number of distinct, differ-ent purposes and the aims can be divided into three main groups: • data description (explorative data structure modelling of any ge-neric n-dimensional data matrix, PCA for example); • regression and prediction (PLS); • classification and prediction of class belongings for new samples (LDA and PLS-DA and ECVA). The aim of this PhD thesis was to verify the possibility of identify-ing and classifying plants or foodstuffs, in different classes, based on the concerted variation in metabolite levels, detected by NMR spectra and using the multivariate data analysis as a tool to inter-pret NMR information. It is important to underline that the results obtained are useful to point out the metabolic consequences of a specific modification on foodstuffs, avoiding the use of a targeted analysis for the different metabolites. The data analysis is performed by applying chemomet-ric multivariate techniques to the NMR dataset of spectra acquired. The research work presented in this thesis is the result of a three years PhD study. This thesis reports the main results obtained from these two main activities: A1) Evaluation of a data pre-processing system in order to mini-mize unwanted sources of variations, due to different instrumental set up, manual spectra processing and to sample preparations arte-facts; A2) Application of multivariate chemiometric models in data analy-sis.

Researches on berry composition in red grape: agronomical, biochemical and molecular approaches

It is well known that the best grape quality can occur only through the achievement of optimal source/sink ratio. Vine balance is in fact a key parameter in controlling berry sugar, acidity and secondary metabolites content (Howell, 2001; Vanden Heuvel et al., 2004). Despite yield reduction and quality improvement are not always strictly related, cluster thinning is considered a technique which could lead to improvement in grape sugar and anthocyanin composition (Dokoozlian and Hirschfelt, 1995; Guidoni et al., 2002). Among several microclimatic variables which may impact grape composition, the effect of cluster light exposure and temperature, which probably act in synergistic and complex way, has been widely explored showing positive even sometimes contradictory results (Spayd et al., 2001; Tarara et al., 2008). Pre-bloom and véraison defoliation are very efficient techniques in inducing cluster microclimatic modification. Furthermore pre-bloom defoliation inducing a lower berry set percentage On these basis the aim of the first experiment of the thesis was to verify in cv Sangiovese the effects on ripening and berry composition of management techniques which may increase source/sink ratio and /or promote light incidence on berries throughout grape ripening. An integrated agronomic, biochemical and microarray approach, aims to understand which mechanisms are involved in berry composition and may be conditioned in the berries during ripening in vines submitted to three treatments. In particular the treatments compared were: a) cluster thinning (increasing in source/sink ratio) b) leaf removal at véraison (increasing cluster light exposure) c) pre-bloom defoliation (increasing source sink ratio and cluster light exposure). Vine response to leaf removal at véraison was further evaluated in the second experiment on three different varieties (Cabernet Sauvignon, Nero d’Avola, Raboso Piave) chosen for their different genetic traits in terms of anthocyanin amount and composition. The integrated agronomic, biochemical and microarray approach, employed in order to understand those mechanisms involved in berry composition of Sangiovese vines submitted to management techniques which may increase source/sink ratio and induce microclimatic changes, bring to interesting results. This research confirmed the main role of source/sink ratio in conditioning sugars metabolism and revealed also that carbohydrates availability is a crucial issue in triggering anthocyanin biosynthesis. More complex is the situation of pre-bloom defoliation, where source/sink and cluster light increase effects are associated to determine final berry composition. It results that the application of pre-bloom defoliation may be risky, as too much dependent on seasonal conditions (rain and temperature) and physiological vine response (leaf area recovery, photosynthetic compensation, laterals regrowth). Early induced stress conditions could bring cluster at véraison in disadvantage to trigger optimal berry ripening processes compared to untreated vines. This conditions could be maintained until harvest, if no previously described physiological recovery occurs. Certainly, light exposure increase linked to defoliation treatments, showed a positive and solid effect on flavonol biosynthesis, as in our conditions temperature was not so different among treatments. Except the last aspects, that could be confirmed also for véraison defoliation, microclimatic changes by themselves seemed not able to induce any modification in berry composition. Further studies are necessary to understand if the peculiar anthocyanic and flavonols composition detected in véraison defoliation could play important role in both color intensity and stability of wines.

N-Heterocyclic carbene complexes of silver, rhodium and iron: structures, dynamics and catalysis

The research performed in the framework of this Master Thesis has been directly inspired by the recent work of an organometallic research group led by Professor Maria Cristina Cassani on a topic related to the structures, dynamics and catalytic activity of N-heterocyclic carbene-amide rhodium(I) complexes1. A series of [BocNHCH2CH2ImR]X (R = Me, X = I, 1a’; R = Bz, X = Br, 1b’; R = trityl, X = Cl, 1c’) amide-functionalized imidazolium salts bearing increasingly bulky N-alkyl substituents were synthetized and characterized. Subsequently, these organic precursors were employed in the synthesis of silver(I) complexes as intermediate compounds on a way to rhodium(I) complexes [Rh(NBD)X(NHC)] (NHC = 1-(2-NHBoc-ethyl)-3-R-imidazolin-2-ylidene; X = Cl, R = Me (3a’), R = Bz (3b’), R = trityl (3c’); X = I, R = Me (4a’)). VT NMR studies of these complexes revealed a restricted rotation barriers about the metal-carbene bond. However, while the rotation barriers calculated for the complexes in which R = Me, Bz (3a’,b’ and 4a) matched the experimental values, this was not true in the trityl case 3c’, where the experimental value was very similar to that obtained for compound 3b’ and much smaller with respect to the calculated one. In addition, the energy barrier derived for 3c’ from line shape simulation showed a strong dependence on the temperature, while the barriers measured for 3a’,b’ did not show this effect. In view of these results and in order to establish the reasons for the previously found inconsistency between calculated and experimental thermodynamic data, the first objective of this master thesis was the preparation of a series of rhodium(I) complexes [Rh(NBD)X(NHC)] (NHC = 1-benzyl-3-R-imidazolin-2-ylidene; X = Cl, R = Me, Bz, trityl, tBu), containing the benzyl substituent as a chiral probe, followed by full characterization. The second objective of this work was to investigate the catalytic activity of the new rhodium compounds in the hydrosilylation of terminal alkynes for comparison purposes with the reported complexes. Another purpose of this work was to employ the prepared N-heterocyclic ligands in the synthesis of iron(II)-NHC complexes.

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.

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.

Immunohistochemical and Molecular Prognostic/Predictive Markers in Neoplastic Diseases

Traditional morphological examinations are not anymore sufficient for a complete evaluation of tumoral tissue and the use of neoplastic markers is of utmost importance. Neoplastic markers can be classified in: diagnostic, prognostic and predictive markers. Three markers were analyzed. 1) Insulin-like growth factor binding protein 2 (IGFBP2) was immunohistochemically examined in prostatic tissues: 40 radical prostatectomies from hormonally untreated patients with their preoperative biopsies, 10 radical prostatectomies from patients under complete androgen ablation before surgery and 10 simple prostatectomies from patients with bladder outlet obstruction. Results were compared with α-methylacyl-CoA racemase (AMACR). IGFBP2 was expressed in the cytoplasm of untreated adenocarcinomas and, to a lesser extent, in HG-PIN; the expression was markedly lower in patients after complete androgen ablation. AMACR was similarly expressed in both adenocarcinoma and HG-PIN, the level being similar in both lesions; the expression was slightly lower in patients after complete androgen ablation. IGFBP2 may be used a diagnostic marker of prostatic adenocarcinomas. 2) Heparan surface proteoglycan immunohistochemical expression was examined in 150 oral squamous cell carcinomas. Follow up information was available in 93 patients (range: 6-34 months, mean: 19±7). After surgery, chemotherapy was performed in 8 patients and radiotherapy in 61 patients. Multivariate and univariate overall survival analyses showed that high expression of syndecan-1 (SYN-1) was associated with a poor prognosis. In patients treated with radiotherapy, such association was higher. SYN-1 is a prognostic marker in oral squamous cell carcinomas; it may also represent a predictive factor for responsiveness to radiotherapy. 3) EGFR was studied in 33 pulmonary adenocarcinomas with traditional DNA sequencing methods and with two mutation-specific antibodies. Overall, the two antibodies had 61.1% sensitivity and 100% specificity in detecting EGFR mutations. EGFR mutation-specific antibodies may represent a predictive marker to identify patients candidate to tyrosine kinase inhibitors therapy.