Molecular analysis of special type breast carcinomas

The project was developed into three parts: the analysis of p63 isoform in breast tumours; the study of intra-tumour eterogeneicity in metaplastic breast carcinoma; the analysis of oncocytic breast carcinoma. p63 is a sequence-specific DNA-binding factor, homologue of the tumour suppressor and transcription factor p53. The human p63 gene is composed of 15 exons and transcription can occur from two distinct promoters: the transactivating isoforms (TAp63) are generated by a promoter upstream of exon 1, while the alternative promoter located in intron 3 leads to the expression of N-terminal truncated isoforms (ΔNp63). It has been demonstrated that anti-p63 antibodies decorate the majority of squamous cell carcinomas of different organs; moreover tumours with myoepithelial differentiation of the breast show nuclear p63 expression. Two new isoforms have been described with the same sequence as TAp63 and ΔNp63 but lacking exon 4: d4TAp63 and ΔNp73L, respectively. Purpose of the study was to investigate the molecular expression of N-terminal p63 isoforms in benign and malignant breast tissues. In the present study 40 specimens from normal breast, benign lesions, DIN/DCIS, and invasive carcinomas were analyzed by immunohistochemistry and RT-PCR (Reverse Transcriptase-PCR) in order to disclose the patterns of p63 expression. We have observed that the full-length isoforms can be detected in non neoplastic and neoplastic lesions, while the short isoforms are only present in the neoplastic cells of invasive carcinomas. Metaplastic carcinomas of the breast are a heterogeneous group of neoplasms which exhibit varied patterns of metaplasia and differentiation. The existence of such non-modal populations harbouring distinct genetic aberrations may explain the phenotypic diversity observed within a given tumour. Intra-tumour morphological heterogeneity is not uncommon in breast cancer and it can often be appreciated in metaplastic breast carcinomas. Aim of this study was to determine the existence of intra-tumour genetic heterogeneity in metaplastic breast cancers and whether areas with distinct morphological features in a given tumour might be underpinned by distinct patterns of genetic aberrations. 47 cases of metaplastic breast carcinomas were retrieved. Out of the 47 cases, 9 had areas that were of sufficient dimensions to be independently microdissected. Our results indicate that at least some breast cancers are composed of multiple non-modal populations of clonally related cells and provide direct evidence that at least some types of metaplastic breast cancers are composed of multiple non-modal clones harbouring distinct genetic aberrations. Oncocytic tumours represent a distinctive set of lesions with typical granular cytoplasmatic eosinophilia of the neoplastic cells. Only rare example of breast oncocytic carcinomas have been reported in literature and the incidence is probably underestimated. In this study we have analysed 33 cases of oncocytic invasive breast carcinoma of the breast, selected according to morphological and immunohistochemical criteria. These tumours were morphologically classified and studied by immunohistochemistry and aCGH. We have concluded that oncocytic breast carcinoma is a morphologic entity with distinctive ultrastructural and histological features; immunohistochemically is characterized by a luminal profile, it has a frequency of 19.8%, has not distinctive clinical features and, at molecular level, shows a specific constellation of genetic aberration.

Molecular analysis of Sigma-1 receptor modulation of the dopamine transporter

Sigma (σ) receptors are well established as a non-opioid, non-phencyclidine, and haloperidol-sensitive receptor family with its own binding profile and a characteristic distribution in the central nervous system (CNS) as well as in endocrine, immune, and some peripheral tissues. Two σ receptors subtypes, termed σ1 and σ2, have been pharmacologically characterized, but, to date, only the σ1 has also been cloned. Activation of σ1 receptors alter several neurotransmitter systems and dopamine (DA) neurotrasmission has been often shown to constitute an important target of σ receptors in different experimental models; however the exact role of σ1 receptor in dopaminergic neurotransmission remains unclear. The DA transporter (DAT) modulates the spatial and temporal aspects of dopaminergic synaptic transmission and interprer the primary mechanism by wich dopaminergic neurons terminate the signal transmission. For this reason present studies have been focused in understanding whether, in cell models, the human subtype of σ1 (hσ1) receptor is able to directly modulate the human DA transporter (hDAT). In the first part of this thesis, HEK-293 and SH-SY5Y cells were permanently transfected with the hσ1 receptor. Subsequently, they were transfected with another plasmid for transiently expressing the hDAT. The hDAT activity was estimated using the described [3H]DA uptake assay and the effects of σ ligands were evaluated by measuring the uptaken [3H]DA after treating the cells with known σ agonists and antagonists. Results illustrated in this thesis demonstrate that activation of overexpressed hσ1 receptors by (+)-pentazocine, the σ1 agonist prototype, determines an increase of 40% of the extracellular [3H]DA uptake, in comparison to non-treated controls and the σ1 antagonists BD-1047 and NE-100 prevent the positive effect of (+)-pentazocine on DA reuptake DA is likely to be considered a neurotoxic molecule. In fact, when levels of intracellular DA abnormally invrease, vescicles can’t sequester the DA which is metabolized by MAO (A and B) and COMT with consequent overproduction of oxygen reactive species and toxic catabolites. Stress induced by these molecules leads cells to death. Thus, for the second part of this thesis, experiments have been performed in order to investigate functional alterations caused by the (+)-pentazocine-mediated increase of DA uptake; particularly it has been investigated if the increase of intracellular [DA] could affect cells viability. Results obtained from this study demonstrate that (+)-pentazocine alone increases DA cell toxicity in a concentration-dependent manner only in cells co-expressing hσ1 and hDAT and σ1 antagonists are able to revert the (+)-pentazocine-induced increase of cell susceptibility to DA toxicity. In the last part of this thesis, the functional cross-talking between hσ1 receptor and hDAT has been further investigated using confocal microscopy. From the acquired data it could be suggested that, following exposure to (+)-pentazocine, the hσ1 receptors massively translocate towards the plasma membrane and colocalize with the hDATs. However, any physical interaction between the two proteins remains to be proved. In conclusion, the presented study shows for the first time that, in cell models, hσ1 receptors directly modulate the hDAT activity. Facilitation of DA uptake induced by (+)-pentazocine is reflected on the increased cell susceptibility to DA toxicity; these effects are prevented by σ1 selective antagonists. Since numerous compounds, including several drugs of abuse, bind to σ1 receptors and activating them could facilitate the damage of dopaminergic neurons, the reported protective effect showed by σ1 antagonists would represent the pharmacological basis to test these compounds in experimental models of dopaminergic neurodegenerative diseases (i.e. Parkinson’s Disease).

Study of Italian isolates of Alternaria spp.: molecular and morphological characterization and pathogenesis on apple tree

It was decided to carry out a morphological and molecular characterization of the Italian Alternaria isolatescollected from apple , and evaluate their pathogenicity and subsequently combining the data collected. The strain collection (174 isolates) was constructed by collecting material (received from extension service personnel) between June and August of 2007, 2008, and 2009. A Preliminary bioassays were performed on detached plant materials (fruit and leaf wounded and unwounded), belonging to the Golden cultivar, with two different kind of inoculation (conidial suspension and conidial filtrate). Symptoms were monitored daily and a value of pathogenicity score (P.S.) was assigned on the basis of the diameter of the necrotic area that developed. On the basis of the bioassays, the number of isolates to undergo further molecular analysis was restricted to a representative set of single spore strains (44 strains). Morphological characteristics of the colony and sporulation pattern were determined according to previous systematic work on small-spored Alternaria spp. (Pryor and Michaelides, 2002 and Hong et al., 2006). Reference strains (Alternaria alternata, Alternaria tenuissima, Alternaria arborescens and four Japanese strains of Alternaria alternata mali pathotype), used in the study were kindly provided by Prof. Barry Pryor, who allows a open access to his own fungal collection. Molecular characterization was performed combining and comparing different data sets obtained from distinct molecular approach: 1) investigation of specific loci and 2) fingerprinting based on diverse randomly selected polymorphic sites of the genome. As concern the single locus analysis, it was chosen to sequence the EndoPG partial gene and three anonymous region (OPA1-3, OPA2- and OPa10-2). These markers has revealed a powerful tool in the latter systematic works on small-spored Alternaria spp. In fact, as reported in literature small-spored Alternaria taxonomy is complicated due to the inability to resolve evolutionary relationships among the taxa because of the lack of variability in the markers commonly used in fungi systematic. The three data set together provided the necessary variation to establish the phylogenetic relationships among the Italian isolates of Alternaria spp. On Italian strains these markers showed a variable number of informative sites (ranging from 7 for EndoPg to 85 for OPA1-3) and the parsimony analysis produced different tree topologies all concordant to define A. arborescens as a mophyletic clade. Fingerprinting analysis (nine ISSR primers and eight AFLP primers combination) led to the same result: a monophyleic A. arborescens clade and one clade containing both A. tenuissima and the A. alternata strains. This first attempt to characterize Italian Alternaria species recovered from apple produced concordant results with what was already described in a similar phylogenetic study on pistachio (Pryor and Michaelides, 2002), on walnut and hazelnut (Hong et al., 2006), apple (Kang et al., 2002) and citurus (Peever et al., 2004). Together with these studies, this research demonstrates that the three morphological groups are widely distributed and occupy similar ecological niches. Furthermore, this research suggest that these Alternaria species exhibit a similar infection pattern despite the taxonomic and pathogenic differences. The molecular characterization of the pathogens is a fundamental step to understanding the disease that is spreading in the apple orchards of the north Italy. At the beginning the causal agent was considered as Alteraria alternata (Marshall and Bertagnoll, 2006). Their preliminary studies purposed a pathogenic system related to the synthesis of toxins. Experimental data of our bioassays suggest an analogous hypothesis, considering that symptoms could be induced after inoculating plant material with solely the filtrate from pathogenic strains. Moreover, positive PCR reactions using AM-toxin gene specific primers, designed for identification of apple infecting Alternaria pathovar, led to a hypothesis that a host specific toxin (toxins) were involved. It remains an intriguing challenge to discover or not if the agent of the “Italian disease” is the same of the one previously typified as Alternaria mali, casual agent of the apple blotch disease.

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.

Application of FTIR microscopy to cultural heritage materials

Research in art conservation has been developed from the early 1950s, giving a significant contribution to the conservation-restoration of cultural heritage artefacts. In fact, only through a profound knowledge about the nature and conditions of constituent materials, suitable decisions on the conservation and restoration measures can thus be adopted and preservation practices enhanced. The study of ancient artworks is particularly challenging as they can be considered as heterogeneous and multilayered systems where numerous interactions between the different components as well as degradation and ageing phenomena take place. However, difficulties to physically separate the different layers due to their thickness (1-200 µm) can result in the inaccurate attribution of the identified compounds to a specific layer. Therefore, details can only be analysed when the sample preparation method leaves the layer structure intact, as for example the preparation of embedding cross sections in synthetic resins. Hence, spatially resolved analytical techniques are required not only to exactly characterize the nature of the compounds but also to obtain precise chemical and physical information about ongoing changes. This thesis focuses on the application of FTIR microspectroscopic techniques for cultural heritage materials. The first section is aimed at introducing the use of FTIR microscopy in conservation science with a particular attention to the sampling criteria and sample preparation methods. The second section is aimed at evaluating and validating the use of different FTIR microscopic analytical methods applied to the study of different art conservation issues which may be encountered dealing with cultural heritage artefacts: the characterisation of the artistic execution technique (chapter II-1), the studies on degradation phenomena (chapter II-2) and finally the evaluation of protective treatments (chapter II-3). The third and last section is divided into three chapters which underline recent developments in FTIR spectroscopy for the characterisation of paint cross sections and in particular thin organic layers: a newly developed preparation method with embedding systems in infrared transparent salts (chapter III-1), the new opportunities offered by macro-ATR imaging spectroscopy (chapter III-2) and the possibilities achieved with the different FTIR microspectroscopic techniques nowadays available (chapter III-3). In chapter II-1, FTIR microspectroscopy as molecular analysis, is presented in an integrated approach with other analytical techniques. The proposed sequence is optimized in function of the limited quantity of sample available and this methodology permits to identify the painting materials and characterise the adopted execution technique and state of conservation. Chapter II-2 describes the characterisation of the degradation products with FTIR microscopy since the investigation on the ageing processes encountered in old artefacts represents one of the most important issues in conservation research. Metal carboxylates resulting from the interaction between pigments and binding media are characterized using synthesised metal palmitates and their production is detected on copper-, zinc-, manganese- and lead- (associated with lead carbonate) based pigments dispersed either in oil or egg tempera. Moreover, significant effects seem to be obtained with iron and cobalt (acceleration of the triglycerides hydrolysis). For the first time on sienna and umber paints, manganese carboxylates are also observed. Finally in chapter II-3, FTIR microscopy is combined with further elemental analyses to characterise and estimate the performances and stability of newly developed treatments, which should better fit conservation-restoration problems. In the second part, in chapter III-1, an innovative embedding system in potassium bromide is reported focusing on the characterisation and localisation of organic substances in cross sections. Not only the identification but also the distribution of proteinaceous, lipidic or resinaceous materials, are evidenced directly on different paint cross sections, especially in thin layers of the order of 10 µm. Chapter III-2 describes the use of a conventional diamond ATR accessory coupled with a focal plane array to obtain chemical images of multi-layered paint cross sections. A rapid and simple identification of the different compounds is achieved without the use of any infrared microscope objectives. Finally, the latest FTIR techniques available are highlighted in chapter III-3 in a comparative study for the characterisation of paint cross sections. Results in terms of spatial resolution, data quality and chemical information obtained are presented and in particular, a new FTIR microscope equipped with a linear array detector, which permits reducing the spatial resolution limit to approximately 5 µm, provides very promising results and may represent a good alternative to either mapping or imaging systems.

Identification and characterization of the sRNA network in Neisseria meningitidis

Bacterial small regulatory RNAs (sRNAs) are posttranscriptional regulators involved in stress responses. These short non-coding transcripts are synthesised in response to a signal, and control gene expression of their regulons by modulating the translation or stability of the target mRNAs, often in concert with the RNA chaperone Hfq. Characterization of a Hfq knock out mutant in Neisseria meningitidis revealed that it has a pleiotropic phenotype, suggesting a major role for Hfq in adaptation to stresses and virulence and the presence of Hfq-dependent sRNA activity. Global gene expression analysis of regulated transcripts in the Hfq mutant revealed the presence of a regulated sRNA, incorrectly annotated as an open reading frame, which we renamed AniS. The synthesis of this novel sRNA is anaerobically induced through activation of its promoter by the FNR global regulator and through global gene expression analyses we identified at least two predicted mRNA targets of AniS. We also performed a detailed molecular analysis of the action of the sRNA NrrF,. We demonstrated that NrrF regulates succinate dehydrogenase by forming a duplex with a region of complementarity within the sdhDA region of the succinate dehydrogenase transcript, and Hfq enhances the binding of this sRNA to the identified target in the sdhCDAB mRNA; this is likely to result in rapid turnover of the transcript in vivo. In addition, in order to globally investigate other possible sRNAs of N. meningitdis we Deep-sequenced the transcriptome of this bacterium under both standard in vitro and iron-depleted conditions. This analysis revealed genes that were actively transcribed under the two conditions. We focused our attention on the transcribed non-coding regions of the genome and, along with 5’ and 3’ untranslated regions, 19 novel candidate sRNAs were identified. Further studies will be focused on the identification of the regulatory networks of these sRNAs, and their targets.

Metodi biochimici e biomolecolari applicati alla medicina clinica: rendimento della real-time TaqMan PCR per la valutazione della resistenza alla claritromicina in H.pylori e tests fecali nella diagnorstica del carcinoma colo-rettale: M2PK, calprotectina e FOBT

1) Background: The most common methods to evaluate clarithromycin resistance is the E-Test, but is time consuming. Resistance of Hp to clarithromycin is due to point mutations in the 23S rRNA. Eight different point mutations have been related to CH resistance, but the large majority of the clarithromycin resistance depends on three point mutations (A2142C, A2142G and A2143G). A novel PCR-based clarithromycin resistance assays, even on paraffin-embedded biopsy specimens, have been proposed. Aims: to assess clarithromycin resistance detecting these point mutation (E-Test as a reference method);secondly, to investigate relation with MIC values. Methods: Paraffin-embedded biopsies of patients Hp-positive were retrieved. The A2142C, A2142G and A2143G point mutations were detected by molecular analysis after DNA extraction by using a TaqMan real-time PCR. Results: The study enrolled 86 patients: 46 resistant and 40 sensible to CH. The Hp status was evaluated at endoscopy, by rapid urease test (RUT), histology and hp culture. According to real-time PCR, 37 specimens were susceptible to clarithromycin (wild type dna) whilst the remaining 49 specimens (57%) were resistant. A2143G is the most frequent mutation. A2142C always express a resistant phenotype and A2142G leads to a resitant phenotype only if homozigous. 2) Background: Colonoscopy work-load for endoscopy services is increasing due to colorectal cancer prevention. We tested a combination of faecal tests to improve accuracy and prioritize the access to colonoscopy. Methods: we tested a combination of fecal tests (FOBT, M2-PK and calprotectin) in a group of 280 patients requiring colonoscopy. Results: 47 patients had CRC and 85 had advanced adenoma/s at colonoscopy/histology. In case of single test, for CRC detection FOBT was the test with the highest specificity and PPV, M2-PK had the highest sensitivity and higher NPV. Combination was more interesting in term of PPV. And the best combination of tests was i-FOBT + M2-PK.

MetaVaccinology: a new vaccine discovery tool

In the last decade, the reverse vaccinology approach shifted the paradigm of vaccine discovery from conventional culture-based methods to high-throughput genome-based approaches for the development of recombinant protein-based vaccines against pathogenic bacteria. Besides reaching its main goal of identifying new vaccine candidates, this new procedure produced also a huge amount of molecular knowledge related to them. In the present work, we explored this knowledge in a species-independent way and we performed a systematic in silico molecular analysis of more than 100 protective antigens, looking at their sequence similarity, domain composition and protein architecture in order to identify possible common molecular features. This meta-analysis revealed that, beside a low sequence similarity, most of the known bacterial protective antigens shared structural/functional Pfam domains as well as specific protein architectures. Based on this, we formulated the hypothesis that the occurrence of these molecular signatures can be predictive of possible protective properties of other proteins in different bacterial species. We tested this hypothesis in Streptococcus agalactiae and identified four new protective antigens. Moreover, in order to provide a second proof of the concept for our approach, we used Staphyloccus aureus as a second pathogen and identified five new protective antigens. This new knowledge-driven selection process, named MetaVaccinology, represents the first in silico vaccine discovery tool based on conserved and predictive molecular and structural features of bacterial protective antigens and not dependent upon the prediction of their sub-cellular localization.

Agenti di encefalopatie spongiformi trasmissibili e zoonosi: tipizzazione molecolare

Transmissible spongiform encephalopathies (TSE) are neurodegenerative diseases caused by the conversion of the host-encoded cellular protein (PrPC) to a disease-associated isoform (PrPSc). The agent responsible for prion diseases may exist as different strains with specific biological and biochemical properties. According to the protein-only hypothesis, prion strain diversity is enciphered in PrPSc conformation. Molecular strain typing methods are based on the electrophoretic mobility of protease resistant core of PrPSc, on the susceptibility to protease digestion, on the glycosylation profile of PrPres and on the conformational stability of PrPSc. In this study a new conformational stability assay was developed based on the differential solubility of PrPC and PrPSc: CSSA (conformational stability and solubility assay). The conformational stability assay was performed by measuring PrPSc solubility in homogenates treated with increasing concentrations of GdnHCl, in the absence of proteinase K. Indeed, dose-response curves allowed estimation of the concentration of GdnHCl able to solubilise 50% of PrPSc. The results showed that this method is valuable for the biochemical typing of strains in bank voles and it is also a promising tool for molecular analysis of natural prion isolates. CSSA also revealed strain-specific PrPSc conformational stabilities of ovine natural isolates so that this feature, combined with the N-terminal PrPSc cleavage, allowed differentiation of classical scrapie, including CH1641-like, from natural goat BSE and experimental sheep BSE. In view of the implications concerning strain similarity between animal and human TSEs, the physico-chemical properties of the Nor98 with two human prion diseases (VPSPr and GSS) were compared in order to investigate the extent of the similarity between animal and human prion strains. The results showed an unexpected heterogeneity of the molecular features among human and sheep TSEs associated with internal PrPres fragments with the possible exception of Nor98 and a case of GSS P102L. These similarities and differences need further investigation by N- and C-terminal sequencing and biological characterization.

A novel specific genetic translocation in epithelioid hemangioendotelioma, showing a fusion of the WWTR1 and CAMTA1 genes, supports the monoclonality of multifocal epithelioid hemangioendotelioma.

Like other vascular tumors, epithelioid hemangioendothelioma (EHE) is multifocal in approximately 50% of cases, and it is unclear whether the separate lesions represent multifocal disease or metastases. We hypothesized that the identification of an identical WWTR1-CAMTA1 rearrangement in different EHEs from the same patient supports the monoclonal origin of EHE. To test our hypothesis, we undertook a molecular analysis of two multicentric EHEs of the liver, including separate tumor samples from each patient. Matherial and Methods: We retrieved two cases of EHE with available tissue for molecular analysis. In both cases, fluorescence in situ hybridization (FISH) was performed to identify the presence of the WWTR1-CAMTA1 rearrangement to confirm the histologic diagnosis of EHE, as previously described. The reverse transcription-polymerase chain reaction (RT-PCR) products were analyzed by electrophoresis and the RT-PCR–amplified products were sequenced using the Sanger method. Results: FISH analysis revealed signal abnormalities in both WWTR1 and CAMTA1. Combined results confirmed the presence of the t(1;3)(1p36.23;3q25.1) translocation in both cases of EHE. Using RT-PCR analysis, we found that the size of the rearranged bands was identical in the different tumors from each patient. The sequence of the fusion gene confirmed a different WWTR1-CAMTA1 rearrangement in each patient, but an identical WWTR1-CAMTA1 rearrangement in the different lesions from each patient. Discussion: Because of its generally indolent clinical course, EHE is commonly classified as a multifocal, rather than metastatic, disease. In this study, we examined two cases of multifocal liver EHE and found an identical WWTR1-CAMTA1 rearrangement in each lesion from the same patient, but not between the two patients. These findings suggest that multifocal EHE arises from metastasis of the same neoplastic clone rather than from the simultaneous formation of multiple neoplastic clones, which supports the monoclonal origin of multifocal EHE.

Analisi molecolare dei linfomi cutanei aggressivi

I linfomi primitivi cutanei riconosciuti nella classificazione della WHO/EORTC si presentano come “entità cliniche distinte” su base clinica, morfologica, immunofenotipica e molecolare. Il fenotipo linfocitario T helper CD4+ caratterizza i CTCL, ma alcune entità a prognosi aggressiva presentano un immunofenotipo citotossico CD8+. Numerosi studi di citogenetica (CGH) e gene-expression profiling (GEP) sono stati condotti negli ultimi anni sui CTCL e sono state riscontrate numerose aberrazioni cromosomiche correlate ai meccanismi di controllo del ciclo cellulare. Scopo del nostro studio è la valutazione delle alterazioni genomiche coinvolte nella tumorigenesi di alcuni CTCL aggressivi: il linfoma extranodale NK/T nasal-type, il linfoma primitivo cutaneo aggressivo epidermotropo (AECTCL) e il gruppo dei PTCL/NOS pleomorfo CD8+. Il materiale bioptico dei pazienti è stato sottoposto alla metodica dell’array-CGH per identificare le anomalie cromosomiche; in alcuni casi di AECTCL è stata applicata la GEP, che evidenzia il profilo di espressione genica delle cellule neoplastiche. I dati ottenuti sono stati valutati in modo statistico, evidenziando le alterazioni cromosomiche comuni significative di ogni entità. In CGH, sono state evidenziate alcune aberrazioni comuni fra le entità studiate, la delezione di 9p21.3, l’amplificazione di 17q, 19p13, 19q13.11-q13.32 , 12q13 e 16p13.3, che determinano la delezione dei geni CDKN2A e CDKN2B e l’attivazione del JAK/STAT signaling pathway. Altre alterazioni definiscono l’amplificazione di c-MYC (8q24) e CCND1/CDK4-6 (11q13). In particolare, sono state evidenziate numerose anomalie genomiche comuni in casi di AECTCL e PTCL/NOS pleomorfo. L’applicazione della GEP in 5 casi di AECTCL ha confermato l’alterata espressione dei geni CDKN2A, JAK3 e STAT6, che potrebbero avere un ruolo diretto nella linfomagenesi. Lo studio di un numero maggiore di casi in GEP e l’introduzione delle nuove indagini molecolari come l’analisi dei miRNA, della whole-exome e whole genome sequences consentiranno di evidenziare alterazioni molecolari correlate con la prognosi, definendo anche nuovi target terapeutici.

New approach in the diagnosis and therapy of hyperphenylalaninemia

Background. Phenylketonuria is the most prevalent inborn error of aminoacid metabolism. Is an autosomal recessive disorder. It results from mutations in the phenylalanine hydroxilase (PAH) gene. Phenotypes can vary from mild hyperphenylalaninemia to a severe phenylketonuria wich, if untreated, results in severe mental retardation. Thanks to neonatal screening programmes, early detection and promp dietetic intervention (phenylalanine restricted diet lifelong) has allowed to avoid neurocognitive complications. Recently, a new therapy is become widely used: the oral supplementation with the PAH cofactor (BH4), wich can alleviate the diet burden. Genotype-phenotype correlation is a reliable tool to predict metabolic phenotype in order to establish a better tailored diet and to assess the potential responsiveness to BH4 therapy. Aim Molecular analysis of the PAH gene, evaluation of genotype-phenotype correlation and prediction of BH4 responsiveness in a group of HPA patients living in Emilia Romagna. Patients and methods. We studied 48 patients affected by PAH deficiency in regular follow-up to our Metabolic Centre. We performed the molecular analysis of these patients using genomic DNA extracted from peripheral blood samples Results. We obtained a full genotipic characterization of 46 patients. We found 87 mutant alleles and 35 different mutations, being the most frequent IVS10-11 G>A (19.3%), R261Q (9.1%), R158Q (9.1%), R408Q (6.8%) and A403V (5.7%), including 2 new ones (L287, N223Y) ever described previously. Notably, we found 15 mutations already identified in BH4-responsive patients, according to the literature. We found 42 different genotipic combinations, most of them in single patients and involving a BH4-responsive mutation. Conclusion. BH4 responsiveness is shown by a consistent number of PAH deficient hyperphenylalaninemic patients. This treatment, combined with a less restricted diet or as monotherapy, can reduce nutritional complications and improve the quality of life of these patients.

Streptococcus agalactiae adapts to glucose stress conditions by modulating gene expression profile

Diabetes mellitus is considered a risk factor for Group B Streptococcus (GBS) infections. Typically, this pathology is associated to high glucose levels in the bloodstream. Although clinical evidences support this notion, the physiological mechanisms underlying GBS adaptation to such conditions are not yet defined. In the attempt to address this issue, we performed comparative global gene expression analysis of GBS grown under glucose-stress conditions and observed that a number of metabolic and virulence genes was differentially regulated. Of importance, we also demonstrated that by knocking-out the csrRS locus the transcription profile of GBS grown in high-glucose conditions was profoundly affected, with more than a third of glucose-dependent genes, including the virulence factor bibA, found to be controlled by this two-component system. Furthermore, in vitro molecular analysis showed that CsrR specifically binds to the bibA promoter and the phosphorilation increases the affinity of the regulator to this promoter region. Moreover, we demonstrated that CsrR acts as a repressor of bibA expression by binding to its promoter in vivo. In conclusion, this work by elucidating both the response of GBS to pathological glucose conditions and the underlined molecular mechanisms will set the basis for a better understanding of GBS pathogenesis.

Evaluation of Microbial Contamination in Bivalve Mollusks: Epidemiology and Diagnosis

Shellfish are filter-feeding organisms that can accumulate many bacteria and viruses. Considering that depuration procedures are not effective in removal of certain microorganisms, shellfish-borne diseases are frequent in many parts of the world, and their control must rely primarily on investigation of prevalence of human pathogens in shellfish and water environment. However, the diffusion of enteric viruses and Vibrio bacteria is not known in many geographical areas, for example in Sardinia, Italy. A survey aimed at investigating the prevalence of Norovirus (NoV), hepatitis A virus (HAV), V. parahaemolyticus, V. cholerae and V. vulnificus was carried out, analyzing both local and imported purified, non-purified and retail shellfish from North Italy and Sardinia. Shellfish from both areas were found contaminated by NoVs, HAV and Vibrio, including retail and purified animals. Molecular analysis evidenced different NoV genogroups and genotypes, including bovine NoVs, as well as pathogenic Vibrio strains, underlining the risk for shellfish consumers. However, also other approaches are needed to control the diffusion of shellfish-borne diseases. It was originally thought that enteric viruses are passively accumulated by shellfish. Recently, it was proven that NoVs bind to specific carbohydrate ligands in oysters, and various NoV strains are characterized by a different bioaccumulation pattern. To deepen the knowledge on this argument, a study was carried out, analyzing bioaccumulation of up to 8 different NoV strains in four different species of shellfish. Different bioaccumulation patterns were observed for each shellfish species and NoV strain used, potentially important in setting up effective shellfish purification protocols. Finally, a novel study of evaluation of viral contamination in shellfish from the French Atlantic coast was carried out following the passage of Xynthia tempest over Western Europe which caused massive destruction. Different enteric viruses were found over a one month period, evidencing the potential of these events of contaminating shellfish.

Analisi molecolare delle fasi evolutive della micosi fungoide

Lo scopo del progetto triennale del dottorato di ricerca è lo studio delle alterazioni genetiche in un gruppo di pazienti affetti da micosi fungoide ed un gruppo di pazienti affetti da sindrome di Sezary. Dalle biopsie cutanee è stato estratto il DNA e analizzato, comparandolo con DNA sano di riferimento, utilizzando la tecnica array-CGH, allo scopo di identificare la presenza di geni potenzialmente implicati nel processo di oncogenesi. Questa analisi è stata eseguita, per ogni paziente, su biopsie effettuate ad una fase iniziale di malattia e ad una fase di progressione della stessa. Sugli stessi pazienti è stata inoltre eseguita un’analisi miRNA. Si ipotizza che il profilo d’espressione dei miRNA possa infatti dare informazioni utili per predire lo stato di malattia, il decorso clinico, la progressione tumorale e la riposta terapeutica. Questo lavoro è stato poi eseguito su biopsie effettuate in pazienti affetti da sindrome di Sezary che, quando non insorge primitivamente come tale, si può considerare una fase evolutiva della micosi fungoide. La valutazione delle alterazioni genetiche, ed in particolare la correlazione esistente tra duplicazione e delezione genetica e sovra/sottoespressione genetica, è stata possibile attraverso l’interpretazione e la comparazione dei dati ottenuti attraverso le tecniche array-CGH e miRNA. Sono stati comparati i risultati ottenuti per valutare quali fossero le alterazioni cromosomiche riscontrate nei diversi stadi di malattia. L’applicazione dell’array-CGH e della metodica di analisi mi-RNA si sono rivelate molto utili per l’identificazione delle diverse aberrazioni cromosomiche presenti nel genoma dei pazienti affetti da micosi fungoide e sindrome di Sezary, per valutare la prognosi del paziente e per cercare di migliorare o trovare nuove linee terapeutiche per il trattamento delle due patologie. Lo studio di questi profili può rappresentare quindi uno strumento di grande importanza nella classificazione e nella diagnosi dei tumori.