Expression of the repressor element-1 silencing transcription factor (REST) is regulated by IGF-I and PKC in human neuroblastoma cells

The repressor element 1-silencing transcription factor (REST) was first identified as a protein that binds to a 21-bp DNA sequence element (known as repressor element 1 (RE1)) resulting in transcriptional repression of the neural-specific genes [Chong et al., 1995; Schoenherr and Anderson, 1995]. The original proposed role for REST was that of a factor responsible for restricting neuronal gene expression to the nervous system by silencing expression of these genes in non-neuronal cells. Although it was initially thought to repress neuronal genes in non-neuronal cells, the role of REST is complex and tissue dependent. In this study I investigated any role played by REST in the induction and patterning of differentiation of SH-SY5Y human neuroblastoma cells exposed to IGF-I. and phorbol 12- myristate 13-acetate (PMA) To down-regulate REST expression we developed an antisense (AS) strategy based on the use of phosphorothioate oligonucleotides (ODNs). In order to evaluate REST mRNA levels, we developed a real-time PCR technique and REST protein levels were evaluated by western blotting. Results showed that nuclear REST is increased in SH-SY5Y neuroblastoma cells cultured in SFM and exposed to IGF-I for 2-days and it then declines in 5-day-treated cells concomitant with a progressive neurite extension. Also the phorbol ester PMA was able to increase nuclear REST levels after 3-days treatment concomitant to neuronal differentiation of neuroblastoma cells, whereas, at later stages, it is down-regulated. Supporting these data, the exposure to PKC inhibitors (GF10923X and Gö6976) and PMA (16nM) reverted the effects observed with PMA alone. REST levels were related to morphological differentiation, expression of growth coneassociated protein 43 (GAP-43; a gene not regulated by REST) and of synapsin I and βIII tubulin (genes regulated by REST), proteins involved in the early stage of neuronal development. We observed that differentiation of SH-SY5Y cells by IGF-I and PMA was accompanied by a significant increase of these neuronal markers, an effect that was concomitant with REST decrease. In order to relate the decreased REST expression with a progressive neurite extension, I investigated any possible involvement of the ubiquitin–proteasome system (UPS), a multienzymatic pathway which degrades polyubiquinated soluble cytoplasmic proteins [Pickart and Cohen, 2004]. For this purpose, SH-SY5Y cells are concomitantly exposed to PMA and the proteasome inhibitor MG132. In SH-SY5Y exposed to PMA and MG 132, we observed an inverse pattern of expression of synapsin I and β- tubulin III, two neuronal differentiation markers regulated by REST. Their cytoplasmic levels are reduced when compared to cells exposed to PMA alone, as a consequence of the increase of REST expression by proteasome inhibitor. The majority of proteasome substrates identified to date are marked for degradation by polyubiquitinylation; however, exceptions to this principle, are well documented [Hoyt and Coffino, 2004]. Interestingly, REST degradation seems to be completely ubiquitin-independent. The expression pattern of REST could be consistent with the theory that, during early neuronal differentiation induced by IGF-I and PKC, it may help to repress the expression of several genes not yet required by the differentiation program and then it declines later. Interestingly, the observation that REST expression is progressively reduced in parallel with cell proliferation seems to indicate that the role of this transcription factor could also be related to cell survival or to counteract apotosis events [Lawinger et al., 2000] although, as shown by AS-ODN experiments, it does not seem to be directly involved in cell proliferation. Therefore, the decline of REST expression is a comparatively later event during maturation of neuroroblasts in vitro. Thus, we propose that REST is regulated by growth factors, like IGF-I, and PKC activators in a time-dependent manner: it is elevated during early steps of neural induction and could contribute to down-regulate genes not yet required by the differentiation program while it declines later for the acquisition of neural phenotypes, concomitantly with a progressive neurite extension. This later decline is regulated by the proteasome system activation in an ubiquitin-indipendent way and adds more evidences to the hypothesis that REST down-regulation contributes to differentiation and arrest of proliferation of neuroblastoma cells. Finally, the glycosylation pattern of the REST protein was analysed, moving from the observation that the molecular weight calculated on REST sequence is about 116 kDa but using western blotting this transcription factor appears to have distinct apparent molecular weight (see Table 1.1): this difference could be explained by post-translational modifications of the proteins, like glycosylation. In fact recently, several studies underlined the importance of O-glycosylation in modulating transcriptional silencing, protein phosphorylation, protein degradation by proteasome and protein–protein interactions [Julenius et al., 2005; Zachara and Hart, 2006]. Deglycosilating analysis showed that REST protein in SH-SY5Y and HEK293 cells is Oglycosylated and not N-glycosylated. Moreover, using several combination of deglycosilating enzymes it is possible to hypothesize the presence of Gal-β(1-3)-GalNAc residues on the endogenous REST, while β(1-4)-linked galactose residues may be present on recombinant REST protein expressed in HEK293 cells. However, the O-glycosylation process produces an immense multiplicity of chemical structures and monosaccharides must be sequentially hydrolyzed by a series of exoglycosidase. Further experiments are needed to characterize all the post-translational modification of the transcription factor REST.

Mechanism of resistance to tyrosine kinase inhibitors in philadelphia-positive acute lymphblastic leukaemia (all): from genetic alterations to impaired RNA editing

The Ph chromosome is the most frequent cytogenetic aberration associated with adult ALL and it represents the single most significant adverse prognostic marker. Despite imatinib has led to significant improvements in the treatment of patients with Ph+ ALL, in the majority of cases resistance developed quickly and disease progressed. Some mechanisms of resistance have been widely described but the full knowledge of contributing factors, driving both the disease and resistance, remains to be defined. The observation of rapid development of lymphoblastic leukemia in mice expressing altered Ikaros (Ik) isoforms represented the background of this study. Ikaros is a zinc finger transcription factor required for normal hemopoietic differentiation and proliferation, particularly in the lymphoid lineages. By means of alternative splicing, Ikaros encodes several proteins that differ in their abilities to bind to a consensus DNA-binding site. Shorter, DNA nonbinding isoforms exert a dominant negative effect, inhibiting the ability of longer heterodimer partners to bind DNA. The differential expression pattern of Ik isoforms in Ph+ ALL patients was analyzed in order to determine if molecular abnormalities involving the Ik gene could associate with resistance to imatinib and dasatinib. Bone marrow and peripheral blood samples from 46 adult patients (median age 55 yrs, 18-76) with Ph+ ALL at diagnosis and during treatment with imatinib (16 pts) or dasatinib (30 pts) were collected. We set up a fast, high-throughput method based on capillary electrophoresis technology to detect and quantify splice variants. 41% Ph+ ALL patients expressed high levels of the non DNA-binding dominant negative Ik6 isoform lacking critical N-terminal zinc-fingers which display abnormal subcellular compartmentalization pattern. Nuclear extracts from patients expressed Ik6 failed to bind DNA in mobility shift assay using a DNA probe containing an Ikaros-specific DNA binding sequence. In 59% Ph+ ALL patients there was the coexistence in the same PCR sample and at the same time of many splice variants corresponded to Ik1, Ik2, Ik4, Ik4A, Ik5A, Ik6, Ik6 and Ik8 isoforms. In these patients aberrant full-length Ikaros isoforms in Ph+ ALL characterized by a 60-bp insertion immediately downstream of exon 3 and a recurring 30-bp in-frame deletion at the end of exon 7 involving most frequently the Ik2, Ik4 isoforms were also identified. Both the insertion and deletion were due to the selection of alternative splice donor and acceptor sites. The molecular monitoring of minimal residual disease showed for the first time in vivo that the Ik6 expression strongly correlated with the BCR-ABL transcript levels suggesting that this alteration could depend on the Bcr-Abl activity. Patient-derived leukaemia cells expressed dominant-negative Ik6 at diagnosis and at the time of relapse, but never during remission. In order to mechanistically demonstrated whether in vitro the overexpression of Ik6 impairs the response to tyrosine kinase inhibitors (TKIs) and contributes to resistance, an imatinib-sensitive Ik6-negative Ph+ ALL cell line (SUP-B15) was transfected with the complete Ik6 DNA coding sequence. The expression of Ik6 strongly increased proliferation and inhibited apoptosis in TKI sensitive cells establishing a previously unknown link between specific molecular defects that involve the Ikaros gene and the resistance to TKIs in Ph+ ALL patients. Amplification and genomic sequence analysis of the exon splice junction regions showed the presence of 2 single nucleotide polymorphisms (SNPs): rs10251980 [A/G] in the exon2/3 splice junction and of rs10262731 [A/G] in the exon 7/8 splice junction in 50% and 36% of patients, respectively. A variant of the rs11329346 [-/C], in 16% of patients was also found. Other two different single nucleotide substitutions not recognized as SNP were observed. Some mutations were predicted by computational analyses (RESCUE approach) to alter cis-splicing elements. In conclusion, these findings demonstrated that the post-transcriptional regulation of alternative splicing of Ikaros gene is defective in the majority of Ph+ ALL patients treated with TKIs. The overexpression of Ik6 blocking B-cell differentiation could contribute to resistance opening a time frame, during which leukaemia cells acquire secondary transforming events that confer definitive resistance to imatinib and dasatinib.

Evaluation of glycoconjugate antigens as vaccine candidates against group A Streptococcus and human immunodeficiency virus infections

This PhD thesis discusses the rationale for design and use of synthetic oligosaccharides for the development of glycoconjugate vaccines and the role of physicochemical methods in the characterization of these vaccines. The study concerns two infectious diseases that represent a serious problem for the national healthcare programs: human immunodeficiency virus (HIV) and Group A Streptococcus (GAS) infections. Both pathogens possess distinctive carbohydrate structures that have been described as suitable targets for the vaccine design. The Group A Streptococcus cell membrane polysaccharide (GAS-PS) is an attractive vaccine antigen candidate based on its conserved, constant expression pattern and the ability to confer immunoprotection in a relevant mouse model. Analysis of the immunogenic response within at-risk populations suggests an inverse correlation between high anti-GAS-PS antibody titres and GAS infection cases. Recent studies show that a chemically synthesized core polysaccharide-based antigen may represent an antigenic structural determinant of the large polysaccharide. Based on GAS-PS structural analysis, the study evaluates the potential to exploit a synthetic design approach to GAS vaccine development and compares the efficiency of synthetic antigens with the long isolated GAS polysaccharide. Synthetic GAS-PS structural analogues were specifically designed and generated to explore the impact of antigen length and terminal residue composition. For the HIV-1 glycoantigens, the dense glycan shield on the surface of the envelope protein gp120 was chosen as a target. This shield masks conserved protein epitopes and facilitates virus spread via binding to glycan receptors on susceptible host cells. The broadly neutralizing monoclonal antibody 2G12 binds a cluster of high-mannose oligosaccharides on the gp120 subunit of HIV-1 Env protein. This oligomannose epitope has been a subject to the synthetic vaccine development. The cluster nature of the 2G12 epitope suggested that multivalent antigen presentation was important to develop a carbohydrate based vaccine candidate. I describe the development of neoglycoconjugates displaying clustered HIV-1 related oligomannose carbohydrates and their immunogenic properties.

Studio molecolare dei meccanismi dell'autoincompatibilità gametofitica in pero europeo (Pyrus communis)

Self-incompatibility (SI) systems have evolved in many flowering plants to prevent self-fertilization and thus promote outbreeding. Pear and apple, as many of the species belonging to the Rosaceae, exhibit RNase-mediated gametophytic self-incompatibility, a widespread system carried also by the Solanaceae and Plantaginaceae. Pear orchards must for this reason contain at least two different cultivars that pollenize each other; to guarantee an efficient cross-pollination, they should have overlapping flowering periods and must be genetically compatible. This compatibility is determined by the S-locus, containing at least two genes encoding for a female (pistil) and a male (pollen) determinant. The female determinant in the Rosaceae, Solanaceae and Plantaginaceae system is a stylar glycoprotein with ribonuclease activity (S-RNase), that acts as a specific cytotoxin in incompatible pollen tubes degrading cellular RNAs. Since its identification, the S-RNase gene has been intensively studied and the sequences of a large number of alleles are available in online databases. On the contrary, the male determinant has been only recently identified as a pollen-expressed protein containing a F-box motif, called S-Locus F-box (abbreviated SLF or SFB). Since F-box proteins are best known for their participation to the SCF (Skp1 - Cullin - F-box) E3 ubiquitine ligase enzymatic complex, that is involved in protein degradation through the 26S proteasome pathway, the male determinant is supposed to act mediating the ubiquitination of the S-RNases, targeting them for the degradation in compatible pollen tubes. Attempts to clone SLF/SFB genes in the Pyrinae produced no results until very recently; in apple, the use of genomic libraries allowed the detection of two F-box genes linked to each S haplotype, called SFBB (S-locus F-Box Brothers). In Japanese pear, three SFBB genes linked to each haplotype were cloned from pollen cDNA. The SFBB genes exhibit S haplotype-specific sequence divergence and pollen-specific expression; their multiplicity is a feature whose interpretation is unclear: it has been hypothesized that all of them participate in the S-specific interaction with the RNase, but it is also possible that only one of them is involved in this function. Moreover, even if the S locus male and female determinants are the only responsible for the specificity of the pollen-pistil recognition, many other factors are supposed to play a role in GSI; these are not linked to the S locus and act in a S-haplotype independent manner. They can have a function in regulating the expression of S determinants (group 1 factors), modulating their activity (group 2) or acting downstream, in the accomplishment of the reaction of acceptance or rejection of the pollen tube (group 3). This study was aimed to the elucidation of the molecular mechanism of GSI in European pear (Pyrus communis) as well as in the other Pyrinae; it was divided in two parts, the first focusing on the characterization of male determinants, and the second on factors external to the S locus. The research of S locus F-box genes was primarily aimed to the identification of such genes in European pear, for which sequence data are still not available; moreover, it allowed also to investigate about the S locus structure in the Pyrinae. The analysis was carried out on a pool of varieties of the three species Pyrus communis (European pear), Pyrus pyrifolia (Japanese pear), and Malus × domestica (apple); varieties carrying S haplotypes whose RNases are highly similar were chosen, in order to check whether or not the same level of similarity is maintained also between the male determinants. A total of 82 sequences was obtained, 47 of which represent the first S-locus F-box genes sequenced from European pear. The sequence data strongly support the hypothesis that the S locus structure is conserved among the three species, and presumably among all the Pyrinae; at least five genes have homologs in the analysed S haplotypes, but the number of F-box genes surrounding the S-RNase could be even greater. The high level of sequence divergence and the similarity between alleles linked to highly conserved RNases, suggest a shared ancestral polymorphism also for the F-box genes. The F-box genes identified in European pear were mapped on a segregating population of 91 individuals from the cross 'Abbé Fétel' × 'Max Red Bartlett'. All the genes were placed on the linkage group 17, where the S locus has been placed both in pear and apple maps, and resulted strongly associated to the S-RNase gene. The linkage with the RNase was perfect for some of the F-box genes, while for others very rare single recombination events were identified. The second part of this study was focused on the research of other genes involved in the SI response in pear; it was aimed on one side to the identification of genes differentially expressed in compatible and incompatible crosses, and on the other to the cloning and characterization of the transglutaminase (TGase) gene, whose role may be crucial in pollen rejection. For the identification of differentially expressed genes, controlled pollinations were carried out in four combinations (self pollination, incompatible, half-compatible and fully compatible cross-pollination); expression profiles were compared through cDNA-AFLP. 28 fragments displaying an expression pattern related to compatibility or incompatibility were identified, cloned and sequenced; the sequence analysis allowed to assign a putative annotation to a part of them. The identified genes are involved in very different cellular processes or in defense mechanisms, suggesting a very complex change in gene expression following the pollen/pistil recognition. The pool of genes identified with this technique offers a good basis for further study toward a better understanding of how the SI response is carried out. Among the factors involved in SI response, moreover, an important role may be played by transglutaminase (TGase), an enzyme involved both in post-translational protein modification and in protein cross-linking. The TGase activity detected in pear styles was significantly higher when pollinated in incompatible combinations than in compatible ones, suggesting a role of this enzyme in the abnormal cytoskeletal reorganization observed during pollen rejection reaction. The aim of this part of the work was thus to identify and clone the pear TGase gene; the PCR amplification of fragments of this gene was achieved using primers realized on the alignment between the Arabidopsis TGase gene sequence and several apple EST fragments; the full-length coding sequence of the pear TGase gene was then cloned from cDNA, and provided a precious tool for further study of the in vitro and in vivo action of this enzyme.

Stem cell pathways in the basal-like breast carcinoma: the role of beta-catenin and HIF-1alpha

Basal-like tumor is an aggressive breast carcinoma subtype that displays an expression signature similar to that of the basal/myoepithelial cells of the breast tissue. Basal-like carcinoma are characterized by over-expression of the Epidermal Growth Factor receptor (EGFR), high frequency of p53 mutations, cytoplasmic/nuclear localization of beta-catenin, overexpression of the Hypoxia inducible factor (HIF)-1alpha target Carbonic Anhydrase isoenzime 9 (CA9) and a gene expression pattern similar to that of normal and cancer stem cells, including the over-expression of the mammary stem cell markers CD44. In this study we investigated the role of p53, EGFR, beta-catenin and HIF-1alpha in the regulation of stem cell features and genes associated with the basal-like gene expression profile. The findings reported in this investigation indicate that p53 inactivation in ductal breast carcinoma cells leads to increased EGFR mRNA and protein levels. In our experimental model, EGFR overexpression induces beta-catenin cytoplasmatic stabilization and transcriptional activity and, by that, leads to increased aggressive features including mammosphere (MS) forming and growth capacity, invasive potential and overexpression of the mammary stem cell gene CD44. Moreover we found that EGFR/beta-catenin axis promotes hypoxia survival in breast carcinoma cells via increased CA9 expression. Indeed beta-catenin positively regulates CA9 expression upon hypoxia exposure. Interestingly we found that beta-catenin inhibits HIF-1alpha transcriptional activity. Looking for the mechanism, we found that CA9 expression is promoted by HIF-1alpha and cytoplasmatic beta-catenin further increased it post-transcriptionally, via direct mRNA binding and stabilization. These data reveal a functional beta-catenin/HIF-1alpha interplay among hallmarks of basal-like tumors and unveil a new functional role for cytoplasmic beta-catenin in the phenotype of such tumors. Therefore it can be proposed that the interplay here described among EGFR/beta-catenin and HIF-1alpha may play a role in breast cancer stem cell survival and function.

Valutazione preclinica di nuovi potenziali bersagli terapeutici per la cura del medulloblastoma.

Il medulloblastoma (MB) è il tumore più comune del sistema nervoso centrale. Attualmente si riesce a curare solo il 50-60% dei pazienti che tuttavia presentano gravi effetti collaterali dovuti alle cure molto aggressive. Una recente classificazione molecolare ha ridistribuito le varianti più comuni di MB in quattro distinti gruppi. In particolare, il gruppo SHH e D sono caratterizzati da un’ alta espressione di MYCN ed associati a prognosi sfavorevole. MYCN è coinvolto nella regolazione della proliferazione cellulare, differenziazione, apoptosi, angiogenesi e metastasi. L’obiettivo di questo lavoro è la valutazione dell’attività antitumorale di oligonucleotidi diretti contro MYCN, sia in vitro su diverse linee cellulari di MB che in vivo in modelli murini xenograft ortotopici di MB. I risultati hanno dimostrato un’ottima inibizione della crescita in linee cellulari di MB, accompagnata da una riduzione della trascrizione genica e dei livelli proteici di MYCN. Inoltre, sono stati confermati tramite RT-PCR alcuni dei geni trovati significativamente variati nell’esperimento di microarray , dopo il trattamento. Molto interessanti sono stati geni quali BIRC5, che risulta down regolato mentre il gene p21 risulta up regolato in tutte le linee cellulari di MB utilizzate. Inoltre, sono stati generati modelli murini di MB utilizzando cellule precedentemente trasfettate per esprimere il gene della luciferasi e valutarne così la crescita tumorale tramite imaging bioluminescente in vivo (BLI), al fine di poter testare l’attività antitumorale data dagli oligonucleotidi. I risultati hanno dimostrato una buona risposta al trattamento come rilevato dalla tecnica di BLI. I dati preliminari prodotti, dimostrano che MYCN potrebbe esser un buon target per la terapia del MB nei casi in cui è overespresso. In particolare, una sua inibizione potrebbe presentare effetti indesiderati moderati in quanto è poco espresso dopo la nascita.

Immunoconiugati contenenti tossine vegetali o siRNA per la deplezione selettiva di cellule leucemiche. Preparazione, citotossicità e studi di mutagenesi sito-specifica.

CD33 is a myeloid cell surface marker absent on normal hematopoietic stem cells and normal tissues but present on leukemic blasts in 90% of adult and paediatric acute myeloid leukaemia (AML) cases. By virtue of its expression pattern and its ability to be rapidly internalized after antibody binding, CD33 has become an attractive target for new immunotherapeutic approaches to treat AML. In this study two immunoconjugates were constructed to contain a humanised single-chain fragment variable antibody (scFv) against CD33 in order to create new antibody-derived therapeutics for AML. The first immunoconjugate was developed to provide targeted delivery of siRNAs as death effectors into leukemic cells. To this purpose, a CD33-specific scFv, modified to include a Cys residue at its C-terminal end (scFvCD33-Cys), was coupled through a disulphide bridge to a nona-d-arginine (9R) peptide carrying a free Cys to the N-terminal. The scFvCD33-9R was able to completely bind siRNAs at a protein to nucleic acid ratio of about 10:1, as confirmed by electrophoretic gel mobility-shift assay. The conjugate was unable to efficiently transduce cytotoxic siRNA (siTox) into the human myeloid cell line U937. We observed slight reductions in cell viability, with a reduction of 25% in comparison to the control group only at high concentration of siTox (300 nM). The second immunoconjugate was constructed by coupling the scFvCD33-Cys to the type 1 ribosome inactivating protein Dianthin 30 (DIA30) through a chemical linking The resulting immunotoxin scFvCD33-DIA30 caused the rapid arrest of protein synthesis, inducing apoptosis and leading ultimately to cell death. In vitro dose-dependent cytotoxicity assays demonstrated that scFvCD33-DIA30 was specifically toxic to CD33-positive cell U937. The concentration needed to reach 50 % of maximum killing efficiency (EC50) was approximately 0.3 nM. The pronounced antigen-restricted cytotoxicity of this novel agent makes it a candidate for further evaluation of its therapeutic potential.

La molecola IRTA1 (immunoglobulin superfamily receptor trans location associated 1) risulta selettivamente espressa nei linfomi non Hodgkin B della zona marginale

La diagnosi di linfoma non Hodgkin B della zona marginale si basa su criteri morfologici e sulla sostanziale negatività per marcatori immunoistochimici espressi in altri sottotipi di linfoma B. L’ obiettivo di questo lavoro è stato, quindi, quello di ricercare una molecola specifica associata ai linfomi della zona marginale. Materiali e Metodi. Sono stati esaminati 2.104 linfomi periferici di entità nosologia eterogenea mediante un anticorpo monoclonale, diretto contro la molecola IRTA1, che riconosce la zona marginale nei tessuti linfoidi umani. Risultati. Si è riscontrata espressione di IRTA1 nel 93% dei linfomi della zona marginale ad insorgenza extranodale e nel 74% di quelli primitivi linfonodali suggerendo la possibilità che questi linfomi possano originare dalle cellule perifollicolari o monocitoidi IRTA1+ riscontrabili nei linfonodi reattivi. La valutazione immunoistochimica mediante doppia colorazione (IRTA1/bcl6), ha inoltre dimostrato come vi sia una modulazione fenotipica nelle cellule marginali neoplastiche nel momento in cui esse colonizzano i follicoli linfoidi e durante la loro circolazione nei centri germinativi. Le cellule marginali neoplastiche che differenziano in senso plasmacellulare perdono l’ espressione di IRTA1 Discussione. In conclusione, tali evidenze hanno permesso di ampliare la conoscenza sulla biologia dei linfomi marginali e sottolineano come IRTA1 sia il primo marcatore diagnostico positivo per queste neoplasie.

Angiogenesis and angioregression gene expression analyses in swine corpus luteum

The corpus luteum (CL) lifespan is characterized by a rapid growth, differentiation and controlled regression of the luteal tissue, accompanied by an intense angiogenesis and angioregression. Indeed, the CL is one of the most highly vascularised tissue in the body with a proliferation rate of the endothelial cells 4- to 20-fold more intense than in some of the most malignant human tumours. This angiogenic process should be rigorously controlled to allow the repeated opportunities of fertilization. After a first period of rapid growth, the tissue becomes stably organized and prepares itself to switch to the phenotype required for its next apoptotic regression. In pregnant swine, the lifespan of the CLs must be extended to support embryonic and foetal development and vascularisation is necessary for the maintenance of luteal function. Among the molecules involved in the angiogenesis, Vascular Endothelial Growth Factor (VEGF) is the main regulator, promoting endothelial cells proliferation, differentiation and survival as well as vascular permeability and vessel lumen formation. During vascular invasion and apoptosis process, the remodelling of the extracellular matrix is essential for the correct evolution of the CL, particularly by the action of specific class of proteolytic enzymes known as matrix metalloproteinases (MMPs). Another important factor that plays a role in the processes of angiogenesis and angioregression during the CL formation and luteolysis is the isopeptide Endothelin-1 (ET-1), which is well-known to be a potent vasoconstrictor and mitogen for endothelial cells. The goal of the present thesis was to study the role and regulation of vascularisation in an adult vascular bed. For this purpose, using a precisely controlled in vivo model of swine CL development and regression, we determined the levels of expression of the members of VEGF system (VEGF total and specific isoforms; VEGF receptor-1, VEGFR-1; VEGF receptor-2, VEGFR-2) and ET- 1 system (ET-1; endothelin converting enzyme-1, ECE-1; endothelin receptor type A, ET-A) as well as the activity of the Ca++/Mg++-dependent endonucleases and gelatinases (MMP-2 and MMP-9). Three experiments were conducted to reach such objectives in CLs isolated from ovaries of cyclic, pregnant or fasted gilts. In the Experiment I, we evaluated the influence of acute fasting on VEGF production and VEGF, VEGFR-2, ET-1, ECE-1 and ET-A mRNA expressions in CLs collected on day 6 after ovulation (midluteal phase). The results indicated a down-regulation of VEGF, VEGFR-2, ET-1 and ECE-1 mRNA expression, although no change was observed for VEGF protein. Furthermore, we observed that fasting stimulated steroidogenesis by luteal cells. On the basis of the main effects of VEGF (stimulation of vessel growth and endothelial permeability) and ET-1 (stimulation of endothelial cell proliferation and vasoconstriction, as well as VEGF stimulation), we concluded that feed restriction possibly inhibited luteal vessel development. This could be, at least in part, compensated by a decrease of vasal tone due to a diminution of ET-1, thus ensuring an adequate blood flow and the production of steroids by the luteal cells. In the Experiment II, we investigated the relationship between VEGF, gelatinases and Ca++/Mg++-dependent endonucleases activities with the functional CL stage throughout the oestrous cycle and at pregnancy. The results demonstrated differential patterns of expression of those molecules in correspondence to the different phases of the oestrous cycle. Immediately after ovulation, VEGF mRNA/protein levels and MMP-9 activity are maximal. On days 5–14 after ovulation, VEGF expression and MMP-2 and -9 activities are at basal levels, while Ca++/Mg++-dependent endonuclease levels increased significantly in relation to day 1. Only at luteolysis (day 17), Ca++/Mg++-dependent endonuclease and MMP-2 spontaneous activity increased significantly. At pregnancy, high levels of MMP-9 and VEGF were observed. These results suggested that during the very early luteal phase, high MMPs activities coupled with high VEGF levels drive the tissue to an angiogenic phenotype, allowing CL growth under LH (Luteinising Hormone) stimulus, while during the late luteal phase, low VEGF and elevate MMPs levels may play a role in the apoptotic tissue and extracellular matrix remodelling during structural luteolysis. In the Experiment III, we described the expression patterns of all distinct VEGF isoforms throughout the oestrous cycle. Furthermore, the mRNA expression and protein levels of both VEGF receptors were also evaluated. Four novel VEGF isoforms (VEGF144, VEGF147, VEGF182, and VEGF164b) were found for the first time in swine and the seven identified isoforms presented four different patterns of expression. All isoforms showed their highest mRNA levels in newly formed CLs (day 1), followed by a decrease during mid-late luteal phase (days 10–17), except for VEGF182, VEGF188 and VEGF144 that showed a differential regulation during late luteal phase (day 14) or at luteolysis (day 17). VEGF protein levels paralleled the most expressed and secreted VEGF120 and VEGF164 isoforms. The VEGF receptors mRNAs showed a different pattern of expression in relation to their ligands, increasing between day 1 and 3 and gradually decreasing during the mid-late luteal phase. The differential regulation of some VEGF isoforms principally during the late luteal phase and luteolysis suggested a specific role of VEGF during tissue remodelling process that occurs either for CL maintenance in case of pregnancy or for noncapillary vessel development essential for tissue removal during structural luteolysis. In summary, our findings allow us to determine relationships among factors involved in the angiogenesis and angioregression mechanisms that take place during the formation and regression of the CL. Thus, CL provides a very interesting model for studying such factors in different fields of the basic research.

New approaches to open problems in gene expression microarray data

In the past decade, the advent of efficient genome sequencing tools and high-throughput experimental biotechnology has lead to enormous progress in the life science. Among the most important innovations is the microarray tecnology. It allows to quantify the expression for thousands of genes simultaneously by measurin the hybridization from a tissue of interest to probes on a small glass or plastic slide. The characteristics of these data include a fair amount of random noise, a predictor dimension in the thousand, and a sample noise in the dozens. One of the most exciting areas to which microarray technology has been applied is the challenge of deciphering complex disease such as cancer. In these studies, samples are taken from two or more groups of individuals with heterogeneous phenotypes, pathologies, or clinical outcomes. these samples are hybridized to microarrays in an effort to find a small number of genes which are strongly correlated with the group of individuals. Eventhough today methods to analyse the data are welle developed and close to reach a standard organization (through the effort of preposed International project like Microarray Gene Expression Data -MGED- Society [1]) it is not unfrequant to stumble in a clinician's question that do not have a compelling statistical method that could permit to answer it.The contribution of this dissertation in deciphering disease regards the development of new approaches aiming at handle open problems posed by clinicians in handle specific experimental designs. In Chapter 1 starting from a biological necessary introduction, we revise the microarray tecnologies and all the important steps that involve an experiment from the production of the array, to the quality controls ending with preprocessing steps that will be used into the data analysis in the rest of the dissertation. While in Chapter 2 a critical review of standard analysis methods are provided stressing most of problems that In Chapter 3 is introduced a method to adress the issue of unbalanced design of miacroarray experiments. In microarray experiments, experimental design is a crucial starting-point for obtaining reasonable results. In a two-class problem, an equal or similar number of samples it should be collected between the two classes. However in some cases, e.g. rare pathologies, the approach to be taken is less evident. We propose to address this issue by applying a modified version of SAM [2]. MultiSAM consists in a reiterated application of a SAM analysis, comparing the less populated class (LPC) with 1,000 random samplings of the same size from the more populated class (MPC) A list of the differentially expressed genes is generated for each SAM application. After 1,000 reiterations, each single probe given a "score" ranging from 0 to 1,000 based on its recurrence in the 1,000 lists as differentially expressed. The performance of MultiSAM was compared to the performance of SAM and LIMMA [3] over two simulated data sets via beta and exponential distribution. The results of all three algorithms over low- noise data sets seems acceptable However, on a real unbalanced two-channel data set reagardin Chronic Lymphocitic Leukemia, LIMMA finds no significant probe, SAM finds 23 significantly changed probes but cannot separate the two classes, while MultiSAM finds 122 probes with score >300 and separates the data into two clusters by hierarchical clustering. We also report extra-assay validation in terms of differentially expressed genes Although standard algorithms perform well over low-noise simulated data sets, multi-SAM seems to be the only one able to reveal subtle differences in gene expression profiles on real unbalanced data. In Chapter 4 a method to adress similarities evaluation in a three-class prblem by means of Relevance Vector Machine [4] is described. In fact, looking at microarray data in a prognostic and diagnostic clinical framework, not only differences could have a crucial role. In some cases similarities can give useful and, sometimes even more, important information. The goal, given three classes, could be to establish, with a certain level of confidence, if the third one is similar to the first or the second one. In this work we show that Relevance Vector Machine (RVM) [2] could be a possible solutions to the limitation of standard supervised classification. In fact, RVM offers many advantages compared, for example, with his well-known precursor (Support Vector Machine - SVM [3]). Among these advantages, the estimate of posterior probability of class membership represents a key feature to address the similarity issue. This is a highly important, but often overlooked, option of any practical pattern recognition system. We focused on Tumor-Grade-three-class problem, so we have 67 samples of grade I (G1), 54 samples of grade 3 (G3) and 100 samples of grade 2 (G2). The goal is to find a model able to separate G1 from G3, then evaluate the third class G2 as test-set to obtain the probability for samples of G2 to be member of class G1 or class G3. The analysis showed that breast cancer samples of grade II have a molecular profile more similar to breast cancer samples of grade I. Looking at the literature this result have been guessed, but no measure of significance was gived before.

Multi-level models and infrastructures for simulating biological system development

The hierarchical organisation of biological systems plays a crucial role in the pattern formation of gene expression resulting from the morphogenetic processes, where autonomous internal dynamics of cells, as well as cell-to-cell interactions through membranes, are responsible for the emergent peculiar structures of the individual phenotype. Being able to reproduce the systems dynamics at different levels of such a hierarchy might be very useful for studying such a complex phenomenon of self-organisation. The idea is to model the phenomenon in terms of a large and dynamic network of compartments, where the interplay between inter-compartment and intra-compartment events determines the emergent behaviour resulting in the formation of spatial patterns. According to these premises the thesis proposes a review of the different approaches already developed in modelling developmental biology problems, as well as the main models and infrastructures available in literature for modelling biological systems, analysing their capabilities in tackling multi-compartment / multi-level models. The thesis then introduces a practical framework, MS-BioNET, for modelling and simulating these scenarios exploiting the potential of multi-level dynamics. This is based on (i) a computational model featuring networks of compartments and an enhanced model of chemical reaction addressing molecule transfer, (ii) a logic-oriented language to flexibly specify complex simulation scenarios, and (iii) a simulation engine based on the many-species/many-channels optimised version of Gillespie’s direct method. The thesis finally proposes the adoption of the agent-based model as an approach capable of capture multi-level dynamics. To overcome the problem of parameter tuning in the model, the simulators are supplied with a module for parameter optimisation. The task is defined as an optimisation problem over the parameter space in which the objective function to be minimised is the distance between the output of the simulator and a target one. The problem is tackled with a metaheuristic algorithm. As an example of application of the MS-BioNET framework and of the agent-based model, a model of the first stages of Drosophila Melanogaster development is realised. The model goal is to generate the early spatial pattern of gap gene expression. The correctness of the models is shown comparing the simulation results with real data of gene expression with spatial and temporal resolution, acquired in free on-line sources.