Bioinformatic methods in applied genomic research

Here I will focus on three main topics that best address and include the projects I have been working in during my three year PhD period that I have spent in different research laboratories addressing both computationally and practically important problems all related to modern molecular genomics. The first topic is the use of livestock species (pigs) as a model of obesity, a complex human dysfunction. My efforts here concern the detection and annotation of Single Nucleotide Polymorphisms. I developed a pipeline for mining human and porcine sequences. Starting from a set of human genes related with obesity the platform returns a list of annotated porcine SNPs extracted from a new set of potential obesity-genes. 565 of these SNPs were analyzed on an Illumina chip to test the involvement in obesity on a population composed by more than 500 pigs. Results will be discussed. All the computational analysis and experiments were done in collaboration with the Biocomputing group and Dr.Luca Fontanesi, respectively, under the direction of prof. Rita Casadio at the Bologna University, Italy. The second topic concerns developing a methodology, based on Factor Analysis, to simultaneously mine information from different levels of biological organization. With specific test cases we develop models of the complexity of the mRNA-miRNA molecular interaction in brain tumors measured indirectly by microarray and quantitative PCR. This work was done under the supervision of Prof. Christine Nardini, at the “CAS-MPG Partner Institute for Computational Biology” of Shangai, China (co-founded by the Max Planck Society and the Chinese Academy of Sciences jointly) The third topic concerns the development of a new method to overcome the variety of PCR technologies routinely adopted to characterize unknown flanking DNA regions of a viral integration locus of the human genome after clinical gene therapy. This new method is entirely based on next generation sequencing and it reduces the time required to detect insertion sites, decreasing the complexity of the procedure. This work was done in collaboration with the group of Dr. Manfred Schmidt at the Nationales Centrum für Tumorerkrankungen (Heidelberg, Germany) supervised by Dr. Annette Deichmann and Dr. Ali Nowrouzi. Furthermore I add as an Appendix the description of a R package for gene network reconstruction that I helped to develop for scientific usage (http://www.bioconductor.org/help/bioc-views/release/bioc/html/BUS.html).

New molecular approaches to control rhabdomyosarcoma onset and metastasis in preclinical systems

Rhabdomyosarcoma is the most common soft tissue sarcoma of childhood. The aim of this study was to identify molecular events involved in rhabdomyosarcoma onset for the development of new therapeutic approaches against specific molecular targets. BALB-p53neu mice develop pelvic rhabdomyosarcoma and combines the activation of HER-2/neu oncogene with the inactivation of an allele of p53 oncosuppressor gene. Gene expression profiling led to the identification of genes potentially involved in rhabdomyosarcoma genesis and therefore of candidate targets. The pattern of expression of p53, HER-2/neu, CDKN2A/p19ARF and IGF-2 suggested that these alterations might be involved in gender-, site- and strain-specific development of rhabdomyosarcoma. Other genes such as CDKN1A/p21 might be involved. The role of IGF-2, CDKN2A/p19ARF and CDKN1A/p21 in tumor growth was investigated with siRNA in murine rhabdomyosarcoma cells. Silencing of p19ARF and p21 induced inhibition of growth and of migration ability, indicating a possible pro-tumor and pro-metastatic role in rhabdomyosarcoma in absence of p53. In addition the autocrine IGF-2/IGF-1R loop found in early phases of cancer progression strengthens its key role in sustaining rhabdomyosarcoma growth. As rhabdomyosarcoma displays defective myogenic differentiation, a therapeutic approach aimed at enhancing myogenic differentiation of rhabdomyosarcoma cells. Forced expression of myogenin was able to restore myogenic differentiation, significantly reduced cell motility and impaired tumor growth and metastatic spread. IL-4 treatment increased rhabdomyosarcoma cell growth, decreased myogenin expression and promoted migration of cells lacking myogenin. Another approach was based on small kinase inhibitors. Agents specifically targeting members of the HER family (Lapatinib), of the IGF system (NVP-AEW541) or downstream signal transducers (NVP-BEZ235) were investigated in vitro in human rhabdomyosarcoma cell lines as therapeutic anti-tumor and anti-metastatic tools. The major effects were obtained with NVP-BEZ235 treatment that was able to strongly inhibit cell growth in vitro and showed anti-metastatic effects in vivo.

Influence of genetic variants and post transcriptional factors on imatinib transporters as determinants of the pharmacological response

Introduction – Although imatinib (IM) is a recognized gold standard in chronic myeloid leukemia (CML) therapy, resistance has emerged in a significant proportion of patients. Aim – The aim of this study was: (1) to investigate the role of genetic variants in genes encoding for IM transporters, as candidate of IM responsiveness and (2) to test the influence of miRNAs on IM response, focusing on efflux transporters. Methods – As a first step, a panel of polymorphisms (SNPs) was genotyped in a subgroup population of 189 patients enrolled in the Tyrosine Kinase Inhibitor Optimization and Selectivity (TOPS) trial. The association with cytogenetic response and molecular response (MR) was assessed for each SNP. As a second step, an in vitro IM-resistant model (K-562 CML cell line) was established. miRNAs profiles were analyzed using Taqman arrays and in silico search was performed for miRNAs deregulated after IM treatment. mRNA and protein expression were quantified using TaqMan realtime PCR and Western blotting, respectively. Results – (1) Among Caucasian patients, ABCB1 rs60023214 significantly correlated with complete MR (P = 0.005). Concerning SNPs combination in IM uptake transporters, the associations with treatment outcomes were statistically significant for both major and complete MR (P = 0.005 and P = 0.01, respectively). (2) ABCB1 protein was not expressed under any conditions of treatment, differently from ABCG2. Two deregulated miRNAs, namely miR-212 and miR-328, were identified to be inversely correlated with ABCG2 (r2= 0.57; p=0.03 and r2=0.47; p=0.06, respectively). Experiments of loss and gain of function confirmed the functional influence of these miRNAs on ABCG2. Conclusion – The multiple candidate gene approach identified single and combination of SNPs that can be proposed as predictor of IM response. The in vitro study suggested that IM resistance could be mediated by miRNA-dependent mechanism. Further studies are needed to validate these preliminary findings.

Ruolo dei recettori degli estrogeni nella trasduzione del segnale nella neoplasia ovarica

Oestrogen induction of cell proliferation is critical in carcinogenesis of gynaecologic tissues. The effects of oestrogens are mediated by Oestrogen receptor (ER) ERα and ERβ, which are members of the nuclear steroid receptor superfamily. The balance between the ERα/ERβ levels seems critical during carcinogenesis due to their different role in proliferation and apoptosis. SERMs are a class of drugs targeting ERs used especially in the treatment of breast cancer, that despite their usefulness, cause side effects. Therefore, it’s important to develop new active molecules without side effects. In a previous work Andreani et al.(2007) investigated the antitumor activity of a new class of indole-derivatives in 60 different human cancer cell lines. In particular they noted that compound named 3L was able to induce a strong antiproliferative effect in cell lines derived from breast, cervix, ovary ,CNS and colon. The aim of this thesis is to characterize the biological effect in ovarian carcinoma cells (IGROV-1), colon adenocarcinoma cells (HT29), cervix adenocarcinoma cells (HelaS3) and breast cancer cells (MCF7). Among the effect exerted on the other cell lines, the most interesting is the cytostatic effect on IGROV-1. In order to identify the 3L molecular target we monitored the 3L concentration in the IGROV-1 nuclear fractions. The analysis revealed that the drug localizes in the nucleus starting from 6 hrs after treatment, suggesting a nuclear target. The stimulation with oestrogen did not increase the proliferation rate in 3L treated cells, suggesting a possible involvement with oestrogen receptors. Due to the 3L fluorescent properties, we demonstrated a colocalization between the ER and the 3L compound. In particular, a chromatin binding assay revealed the presence of a 3L-ERβ complex bound to DNA, interaction that may be the cause of the observed antiproliferative effect.

Genetic Manipulation, Gene Silencing and Biomarker Development in Multiple Experimental Models.

The thesis is set in three different parts, according to the relative experimental models. First, the domestic pig (Sus scrofa) is part of the study on reproductive biotechnologies: the transgenesis technique of Sperm Mediated Gene Transfer is widely studied starting from the quality of the semen, through the study of multiple uptakes of exogenous DNA and lastly used in the production of multi-transgenic blastocysts. Finally we managed to couple the transgenesis pipeline with sperm sorting and therefore produced transgenic embryos of predetermined sex. In the second part of the thesis the attention is on the fruit fly (Drosophila melanogaster) and on its derived cell line: the S2 cells. The in vitro and in vivo models are used to develop and validate an efficient way to knock down the myc gene. First an efficient in vitro protocol is described, than we demonstrate how the decrease in myc transcript remarkably affects the ribosome biogenesis through the study of Polysome gradients, rRNA content and qPCR. In vivo we identified two optimal drivers for the conditional silencing of myc, once the flies are fed with RU486: the first one is throughout the whole body (Tubulin), while the second is a head fat body driver (S32). With these results we present a very efficient model to study the role of myc in multiple aspects of translation. In the third and last part, the focus is on human derived lung fibroblasts (hLF-1), mouse tail fibroblasts and mouse tissues. We developed an efficient assay to quantify the total protein content of the nucleus on a single cell level via fluorescence. We coupled the protocol with classical immunofluorescence so to have at the same time general and particular information, demonstrating that during senescence nuclear proteins increase by 1.8 fold either in human cells, mouse cells and mouse tissues.

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.

Molecular approach to Neurodegenerative Disorders: Role of Nociceptin/Orphanin FQ - NOP System in Parkinson and Epigenetic Mechanism in Alzheimer's Disease

With life expectancies increasing around the world, populations are getting age and neurodegenerative diseases have become a global issue. For this reason we have focused our attention on the two most important neurodegenerative diseases: Parkinson’s and Alzheimer’s. Parkinson’s disease is a chronic progressive neurodegenerative movement disorder of multi-factorial origin. Environmental toxins as well as agricultural chemicals have been associated with PD. Has been observed that N/OFQ contributes to both neurotoxicity and symptoms associated with PD and that pronociceptin gene expression is up-regulated in rat SN of 6-OHDA and MPP induced experimental parkinsonism. First, we investigated the role of N/OFQ-NOP system in the pathogenesis of PD in an animal model developed using PQ and/or MB. Then we studied Alzheimer's disease. This disorder is defined as a progressive neurologic disease of the brain leading to the irreversible loss of neurons and the loss of intellectual abilities, including memory and reasoning, which become severe enough to impede social or occupational functioning. Effective biomarker tests could prevent such devastating damage occurring. We utilized the peripheral blood cells of AD discordant monozygotic twin in the search of peripheral markers which could reflect the pathology within the brain, and also support the hypothesis that PBMC might be a useful model of epigenetic gene regulation in the brain. We investigated the mRNA levels in several genes involve in AD pathogenesis, as well DNA methylation by MSP Real-Time PCR. Finally by Western Blotting we assess the immunoreactivity levels for histone modifications. Our results support the idea that epigenetic changes assessed in PBMCs can also be useful in neurodegenerative disorders, like AD and PD, enabling identification of new biomarkers in order to develop early diagnostic programs.

Mappaggio fine di un QTL principale per la resa in granella sul cromosoma 3B di frumento duro

In durum wheat, two major QTL for grain yield (Qyld.idw-2B and Qyld.idw-3B) and related traits were identified in a recombinant population derived from Kofa and Svevo (Maccaferri et al. 2008). To further investigate the genetic and physiological basis of allelic variation for this important trait, the fine mapping of Qyld.idw-2B e Qyld.idw-3B was done during the PhD. In this regard, new molecular markers were added to increase the map resolution in the target interval. For Qyld.idw-2B region COS markers derived from the synteny between wheat and rice/ sorghum /brachypodiu genomes were screened. While for Qyld.idw-3B region SSR, ISBP and COS markers obtained from BAC end-sequences and BAC sequences generated during the construction of the 3B physical map (Paux et al., 2008) were screened. In the RIL population a final map resolution of 2,8 markers/cM for Qyld.idw-2B and 0,6 markers/cM for Qyld.idw-3B were obtained. Eighteen pairs of near-isogenic lines (NILs) for Qyld.idw-3B were obtained from F4:5 heterogeneous inbred families. In order to confirm the phenotypic effect of the QTL all pairs were evaluated in field trials (2010 and 2011) for all traits. Three pairs of NILs, with contrasted haplotypes at the target region, were crossed to produce a large F2 population (ca. 7,500 plants in total) that was screened for the identification of recombinants. A total of 233 homozygous F4:5 segmental isolines were obtained and the phenotypic and genotypic characterization of these materials were done. A fine mapping for Qyld.idw-3B was obtained and the QTL peak was identified in a interval of 0,4 cM. All markers were anchored to the Chinese Spring physical map of chr. 3B, which allowed us to identify the BAC Contigs spanning the QTL region and to assign the QTL peak to Contig 954. Sequencing of this contig has revealed the presence of 42 genes.

Identification of the N-Linked Glycosylation Sites of the Transcription Factor Rest and Effect of Glycosylation on DNA Binding and Transcriptional Activity

REST is a zinc-finger transcription factor implicated in several processes such as maintenance of embryonic stem cell pluripotency and regulation of mitotic fidelity in non-neuronal cells [Chong et al., 1995]. The gene encodes for a 116-kDa protein that acts as a molecular platform for co-repressors recruitment and promotes modifications of DNA and histones [Ballas, 2005]. REST showed different apparent molecular weights, consistent with the possible presence of post-translational modifications [Lee et al., 2000]. Among these the most common is glycosylation, the covalent attachment of carbohydrates during or after protein synthesis [Apweiler et al., 1999] My thesis has ascertained, for the first time, the presence of glycan chians in the transcription factor REST. Through enzymatic deglycosylation and MS, oligosaccharide composition of glycan chains was evaluated: a complex mixture of glycans, composed of N-acetylgalactosamine, galactose and mannose, was observed thus confirming the presence of O- and N-linked glycan chains. Glycosylation site mapping was done using a 18O-labeling method and MS/MS and twelve potential N-glycosylation sites were identified. The most probable glycosylation target residues were mutated through site-directed mutagenesis and REST mutants were expressed in different cell lines. Variations in the protein molecular weight and mutant REST ability to bind the RE-1 sequence were analyzed. Gene reporter assays showed that, altogether, removal of N-linked glycan chains causes loss of transcriptional repressor function, except for mutant N59 which showed a slight residual repressor activity in presence of IGF-I. Taken togheter these results demonstrate the presence of complex glycan chians in the transcription factor REST: I have depicted their composition, started defining their position on the protein backbone and identified their possible role in the transcription factor functioning. Considering the crucial role of glycosylation and transcription factors activity in the aetiology of many diseases, any further knowledge could find important and interesting pharmacological application.

Different addictive drug exposure induces selective alterations of the Endogenous Opioid System : modulation of transcription and epigenetic mechanisms

Drug addiction manifests clinically as compulsive drug seeking, and cravings that can persist and recur even after extended periods of abstinence. The fundamental principle that unites addictive drugs is that each one enhances synaptic DA by means that dissociate it from normal behavioral control, so that they act to reinforce their own acquisition. Our attention has focused on the study of phenomena associated with the consumption of alcohol and heroin. Alcohol has long been considered an unspecific pharmacological agent, recent molecular pharmacology studies have shown that acts on different primary targets. Through gene expression studies conducted recently it has been shown that the classical opioid receptors are differently involved in the consumption of ethanol and, furthermore, the system nociceptin / NOP, included in the family of endogenous opioid system, and both appear able to play a key role in the initiation of alcohol use in rodents. What emerges is that manipulation of the opioid system, nociceptin, may be useful in the treatment of addictions and there are several evidences that support the use of this strategy. The linkage between gene expression alterations and epigenetic modulation in PDYN and PNOC promoters following alcohol treatment confirm the possible chromatin remodeling mechanism already proposed for alcoholism. In the second part of present study, we also investigated alterations in signaling molecules directly associated with MAPK pathway in a unique collection of postmortem brains from heroin abusers. The interest was focused on understanding the effects that prolonged exposure of heroin can cause in an individual, over the entire MAPK cascade and consequently on the transcription factor ELK1, which is regulated by this pathway. We have shown that the activation of ERK1/2 resulting in Elk-1 phosphorylation in striatal neurons supporting the hypothesis that prolonged exposure to substance abuse causes a dysregulation of MAPK pathway.

Sulforaphane as a multifunctional neuroprotective molecule to prevent and slow down the progression of Alzheimer’s disease

Oxidative stress has been implicated in the pathogenesis of a number of diseases including neurodegenerative disorders, cancer, ischemia, etc. Alzheimer’s disease (AD) is histopathologically characterized by the presence of extracellular senile plaque (SP), predominantly consisting of fibrillar amyloid-peptide (Aβ), intracellular neurofibrillary tangles (NFTs), composed of hyperphosphorylated tau protein, and cell loss in the selected regions of the brain. However, the pathogenesis of AD remains largely unknown, but a number of hypothesis were proposed for AD mechanisms, which include: the amyloid cascade, excitotoxicity, oxidative stress and inflammation hypothesis, and all of them are based, to some extent on the role of A. Accumulated evidence indicates that the increased levels of ROS may act as important mediators of synaptic loss and eventually promote formation of neurofibrillary tangles and senile plaques. Therefore a vicious circle between ROS and Aaccumulation may accelerate progression of AD. For these reasons, growing attention has focused on oxidative mechanism of Atoxicity as well as the search for novel neuroprotective agents. A strategy to prevent the oxidative stress in neurons may be the use of chemopreventive agents as inducers of antioxidant and phase 2 enzymes. Sulforaphane (SF), derived from corresponding glucoraphanin, glucosinolate found in abundance in cruciferous vegetables, has recently gained attention as a potential neuroprotective compound inducer of antioxidant phase 2 enzymes. Consistent with this evidence, the study is aimed at identifying the SF ability to prevent and counteract the oxidative damage inducted by oligomers of Aβ (1-42) in terms of impairment in the intracellular redox state and cellular death in differentiated human neuroblastoma and microglia primary cultures. In addition we will evaluated the mechanism underlying the SF neuroprotection activity.

Il coniglio come modello sperimentale per lo studio degli effetti neurologici indotti da Cobalto

Sono stati studiati gli effetti tossici dell’esposizione cronica a cobalto e cromo. In passato, questa tossicità, che colpiva lavoratori esposti per ragioni occupazionali, è stata un problema molto sentito. Tuttavia, recenti pubblicazioni hanno descritto una specifica tossicità mediata da elevati livelli di cobalto e cromo, anche in pazienti portatori di protesi metalliche, quali gli impianti d’anca. Anche se sintomi clinici tra cui, cecità, sordità e neuropatia periferica, suggeriscono uno specifico neurotropismo, ancora poco è conosciuto delle basi neuropatologiche di questo processo ed oltretutto non ne è ancora stata apportata un’evidenza sperimentale. In questo progetto di ricerca, quindi, si è voluto approfondire il meccanismo patogenetico da cui scaturiscono tali sintomi neurologici, utilizzando come modello sperimentale il coniglio. Conigli New Zealand White sono stati trattati con dosi endovenose ripetute di cobalto e cromo, inoculati singolarmente od in associazione tra loro. Nessuna evidente alterazione clinica o patologica è stata associata alla somministrazione di solo cromo, nonostante gli elevati livelli in sangue e tessuti, mentre i trattati con cobalto-cromo o solo cobalto hanno mostrato segni clinici gravanti sul sistema vestibolo-cocleare; il cobalto, quindi, è stato identificato come il maggiore elemento scatenante neurotossicità. Inoltre all’esame istopatologico gli animali hanno mostrato severa deplezione delle cellule gangliari retiniche e cocleari, assieme a danno al nervo ottico e perdita di cellule sensitive capellute dell’orecchio. È risultato infine evidente che la gravità delle alterazioni è stata correlata al dosaggio ed al tempo di esposizione; dati questi che confermano, quindi, le precedenti osservazioni fatte su pazienti umani esposti a rilascio abnorme di cobalto e cromo da usura di protesi d’anca. È stato ipotizzato che il cobalto agisca sui mitocondri provocando l’incremento di produzione di specie reattive dell’ossigeno e il rilascio di fattori proapoptotici, causando sulle cellule neuronali un danno proporzionale al loro fabbisogno energetico e grado di mielinizzazione.

The impact of polymorphisms in P-gp, DNA repair and folic acid metabolism genes in newly diagnosed multiple myeloma patients treated with thalidomide plus dexamethasone, with or without bortezomib

The principle aim of this study was to investigate biological predictors of response and resistance to multiple myeloma treatment. Two hypothesis had been proposed as responsible of responsiveness: SNPs in DNA repair and Folate pathway, and P-gp dependent efflux. As a first objective, panel of SNPs in DNA repair and Folate pathway genes, were analyzed. It was a retrospective study in a group of 454, previously untreated, MM patients enrolled in a randomized phase III open-label study. Results show that some SNPs in Folate pathway are correlated with response to MM treatment. MTR genotype was associated with favorable response in the overall population of MM patients. However, this relation, disappear after adjustment for treatment response. When poor responder includes very good partial response, partial response and stable/progressive disease MTFHR rs1801131 genotype was associated with poor response to therapy. This relation - unlike in MTR – was still significant after adjustment for treatment response. Identification of this genetic variant in MM patients could be used as an independent prognostic factor for therapeutic outcome in the clinical practice. In the second objective, basic disposition characteristics of bortezomib was investigated. We demonstrated that bortezomib is a P-gp substrate in a bi-directional transport study. We obtain apparent permeability rate values that together with solubility values can have a crucial implication in better understanding of bortezomib pharmacokinetics with respect to the importance of membrane transporters. Subsequently, in view of the importance of P-gp for bortezomib responsiveness a panel of SNPs in ABCB1 gene - coding for P-gp - were analyzed. In particular we analyzed five SNPs, none of them however correlated with treatment responsiveness. However, we found a significant association between ABCB1 variants and cytogenetic abnormalities. In particular, deletion of chromosome 17 and t(4;14) translocation were present in patients harboring rs60023214 and rs2038502 variants respectively.