Genomic and non genomic effects of elevated concentration of anabolic steroids in human neuronal cells

Nandrolone and other anabolic androgenic steroids (AAS) at elevated concentration can alter the expression and function of neurotransmitter systems and contribute to neuronal cell death. This effect can explain the behavioural changes, drug dependence and neuro degeneration observed in steroid abuser. Nandrolone treatment (10-8M–10-5M) caused a time- and concentration-dependent downregulation of mu opioid receptor (MOPr) transcripts in SH-SY5Y human neuroblastoma cells. This effect was prevented by the androgen receptor (AR) antagonist hydroxyflutamide. Receptor binding assays confirmed a decrease in MOPr of approximately 40% in nandrolonetreated cells. Treatment with actinomycin D (10-5M), a transcription inhibitor, revealed that nandrolone may regulate MOPr mRNA stability. In SH-SY5Y cells transfected with a human MOPr luciferase promoter/reporter construct, nandrolone did not alter the rate of gene transcription. These results suggest that nandrolone may regulate MOPr expression through post-transcriptional mechanisms requiring the AR. Cito-toxicity assays demonstrated a time- and concentration dependent decrease of cells viability in SH-SY5Y cells exposed to steroids (10-6M–10-4M). This toxic effects is independent of activation of AR and sigma-2 receptor. An increased of caspase-3 activity was observed in cells treated with Nandrolone 10-6M for 48h. Collectively, these data support the existence of two cellular mechanisms that might explain the neurological syndromes observed in steroids abuser.

Genomic characterization of the italian wolf (Canis lupus): the genes involved in black coat colour determination and application of microarray technique for snps detection.

This study provides a comprehensive genetic overview on the endangered Italian wolf population. In particular, it focuses on two research lines. On one hand, we focalised on melanism in wolf in order to isolate a mutation related with black coat colour in canids. With several reported black individuals (an exception at European level), the Italian wolf population constituted a challenging research field posing many unanswered questions. As found in North American wolf, we reported that melanism in the Italian population is caused by a different melanocortin pathway component, the K locus, in which a beta-defensin protein acts as an alternative ligand for the Mc1r. This research project was conducted in collaboration with Prof. Gregory Barsh, Department of Genetics and Paediatrics, Stanford University. On the other hand, we performed analysis on a high number of SNPs thanks to a customized Canine microarray useful to integrate or substitute the STR markers for genotyping individuals and detecting wolf-dog hybrids. Thanks to DNA microchip technology, we obtained an impressive amount of genetic data which provides a solid base for future functional genomic studies. This study was undertaken in collaboration with Prof. Robert K. Wayne, Department of Ecology and Evolutionary Biology, University of California, Los Angeles (UCLA).

Genomic and transcriptional analysis of allergen genes in apple (Malus x domestica)

Apple consumption is highly recomended for a healthy diet and is the most important fruit produced in temperate climate regions. Unfortunately, it is also one of the fruit that most ofthen provoks allergy in atopic patients and the only treatment available up to date for these apple allergic patients is the avoidance. Apple allergy is due to the presence of four major classes of allergens: Mal d 1 (PR-10/Bet v 1-like proteins), Mal d 2 (Thaumatine-like proteins), Mal d 3 (Lipid transfer protein) and Mal d 4 (profilin). In this work new advances in the characterization of apple allergen gene families have been reached using a multidisciplinary approach. First of all, a genomic approach was used for the characterization of the allergen gene families of Mal d 1 (task of Chapter 1), Mal d 2 and Mal d 4 (task of Chapter 5). In particular, in Chapter 1 the study of two large contiguos blocks of DNA sequences containing the Mal d 1 gene cluster on LG16 allowed to acquire many new findings on number and orientation of genes in the cluster, their physical distances, their regulatory sequences and the presence of other genes or pseudogenes in this genomic region. Three new members were discovered co-localizing with the other Mal d 1 genes of LG16 suggesting that the complexity of the genetic base of allergenicity will increase with new advances. Many retrotranspon elements were also retrieved in this cluster. Due to the developement of molecular markers on the two sequences, the anchoring of the physical and the genetic map of the region has been successfully achieved. Moreover, in Chapter 5 the existence of other loci for the Thaumatine-like protein family in apple (Mal d 2.03 on LG4 and Mal d 2.02 on LG17) respect the one reported up to now was demonstred for the first time. Also one new locus for profilins (Mal d 4.04) was mapped on LG2, close to the Mal d 4.02 locus, suggesting a cluster organization for this gene family, as is well reported for Mal d 1 family. Secondly, a methodological approach was used to set up an highly specific tool to discriminate and quantify the expression of each Mal d 1 allergen gene (task of Chapter 2). In aprticular, a set of 20 Mal d 1 gene specific primer pairs for the quantitative Real time PCR technique was validated and optimized. As a first application, this tool was used on leaves and fruit tissues of the cultivar Florina in order to identify the Mal d 1 allergen genes that are expressed in different tissues. The differential expression retrieved in this study revealed a tissue-specificity for some Mal d 1 genes: 10/20 Mal d 1 genes were expressed in fruits and, indeed, probably more involved in the allergic reactions; while 17/20 Mal d 1 genes were expressed in leaves challenged with the fungus Venturia inaequalis and therefore probably interesting in the study of the plant defense mechanism. In Chapter 3 the specific expression levels of the 10 Mal d 1 isoallergen genes, found to be expressed in fruits, were studied for the first time in skin and flesh of apples of different genotypes. A complex gene expression profile was obtained due to the high gene-, tissue- and genotype-variability. Despite this, Mal d 1.06A and Mal d 1.07 expression patterns resulted particularly associated with the degree of allergenicity of the different cultivars. They were not the most expressed Mal d 1 genes in apple but here it was hypotized a relevant importance in the determination of allergenicity for both qualitative and quantitative aspects of the Mal d 1 gene expression levels. In Chapter 4 a clear modulation for all the 17 PR-10 genes tested in young leaves of Florina after challenging with the fungus V. inaequalis have been reported but with a peculiar expression profile for each gene. Interestingly, all the Mal d 1 genes resulted up-regulated except Mal d 1.10 that was down-regulated after the challenging with the fungus. The differences in direction, timing and magnitude of induction seem to confirm the hypothesis of a subfunctionalization inside the gene family despite an high sequencce and structure similarity. Moreover, a modulation of PR-10 genes was showed both in compatible (Gala-V. inaequalis) and incompatible (Florina-V. inaequalis) interactions contribute to validate the hypothesis of an indirect role for at least some of these proteins in the induced defense responses. Finally, a certain modulation of PR-10 transcripts retrieved also in leaves treated with water confirm their abilty to respond also to abiotic stress. To conclude, the genomic approach used here allowed to create a comprehensive inventory of all the genes of allergen families, especially in the case of extended gene families like Mal d 1. This knowledge can be considered a basal prerequisite for many further studies. On the other hand, the specific transcriptional approach make it possible to evaluate the Mal d 1 genes behavior on different samples and conditions and therefore, to speculate on their involvement on apple allergenicity process. Considering the double nature of Mal d 1 proteins, as apple allergens and as PR-10 proteins, the gene expression analysis upon the attack of the fungus created the base for unravel the Mal d 1 biological functions. In particular, the knowledge acquired in this work about the PR-10 genes putatively more involved in the specific Malus-V. inaequalis interaction will be helpful, in the future, to drive the apple breeding for hypo-allergenicity genotype without compromise the mechanism of response of the plants to stress conditions. For the future, the survey of the differences in allergenicity among cultivars has to be be thorough including other genotypes and allergic patients in the tests. After this, the allelic diversity analysis with the high and low allergenic cultivars on all the allergen genes, in particular on the ones with transcription levels correlated to allergencity, will provide the genetic background of the low ones. This step from genes to alleles will allow the develop of molecular markers for them that might be used to effectively addressed the apple breeding for hypo-allergenicity. Another important step forward for the study of apple allergens will be the use of a specific proteomic approach since apple allergy is a multifactor-determined disease and only an interdisciplinary and integrated approach can be effective for its prevention and treatment.

Design and fabrication of MOMS-based ultrasonic probes for minimally invasive endoscopic applications

A Micro-opto-mechanical systems (MOMS) based technology for the fabrication of ultrasonic probes on optical fiber is presented. Thanks to the high miniaturization level reached, the realization of an ultrasonic system constituted by ultrasonic generating and detecting elements, suitable for minimally invasive applications or Non Destructive Evaluation (NDE) of materials at high resolution, is demonstrated. The ultrasonic generation is realized by irradiating a highly absorbing carbon film patterned on silicon micromachined structures with a nanosecond pulsed laser source, generating a mechanical shock wave due to the thermal expansion of the film induced by optical energy conversion into heat. The short duration of the pulsed laser, together with an appropriate emitter design, assure high frequency and wide band ultrasonic generation. The acoustic detection is also realized on a MOMS device using an interferometric receiver, fabricated with a Fabry-Perot optical cavity realized by means of a patterned SU-8 and two Al metallization levels. In order to detect the ultrasonic waves, the cavity is interrogated by a laser beam measuring the reflected power with a photodiode. Various issues related to the design and fabrication of these acoustic probes are investigated in this thesis. First, theoretical models are developed to characterize the opto-acoustic behavior of the devices and estimate their expected acoustic performances. Tests structures are realized to derive the relevant physical parameters of the materials constituting the MOMS devices and determine the conditions theoretically assuring the best acoustic emission and detection performances. Moreover, by exploiting the models and the theoretical results, prototypes of acoustic probes are designed and their fabrication process developed by means of an extended experimental activity.

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).

Depicting the role of CDKN2A/ARF alterations in adult BCR-ABL1-positive acute lymphoblastic leukemia patients: from genomic deletions to prognostic impact

This 9p21 locus, encode for important proteins involved in cell cycle regulation and apoptosis containing the p16/CDKN2A (cyclin-dependent kinase inhibitor 2a) tumor suppressor gene and two other related genes, p14/ARF and p15/CDKN2B. This locus, is a major target of inactivation in the pathogenesis of a number of human tumors, both solid and haematologic, and is a frequent site of loss or deletion also in acute lymphoblastic leukemia (ALL) ranging from 18% to 45% 1. In order to explore, at high resolution, the frequency and size of alterations affecting this locus in adult BCR-ABL1-positive ALL and to investigate their prognostic value, 112 patients (101 de novo and 11 relapse cases) were analyzed by genome-wide single nucleotide polymorphisms arrays and gene candidate deep exon sequencing. Paired diagnosis-relapse samples were further available and analyzed for 19 (19%) cases. CDKN2A/ARF and CDKN2B genomic alterations were identified in 29% and 25% of newly diagnosed patients, respectively. Deletions were monoallelic in 72% of cases and in 43% the minimal overlapping region of the lost area spanned only the CDKN2A/2B gene locus. The analysis at the time of relapse showed an almost significant increase in the detection rate of CDKN2A/ARF loss (47%) compared to diagnosis (p = 0.06). Point mutations within the 9p21 locus were found at very low level with only a non-synonymous substition in the exon 2 of CDKN2A. Finally, correlation with clinical outcome showed that deletions of CDKN2A/B are significantly associated with poor outcome in terms of overall survival (p = 0.0206), disease free-survival (p = 0.0010) and cumulative incidence of relapse (p = 0.0014). The inactivation of 9p21 locus by genomic deletions is a frequent event in BCR-ABL1-positive ALL. Deletions are frequently acquired at the leukemia progression and work as a poor prognostic marker.

Early-type galaxies as probes of galaxy formation and cosmology

The goal of this thesis is to analyze the possibility of using early-type galaxies to place evolutionary and cosmological constraints, by both disentangling what is the main driver of ETGs evolution between mass and environment, and developing a technique to constrain H(z) and the cosmological parameters studying the ETGs age-redshift relation. The (U-V) rest-frame color distribution is studied as a function of mass and environment for two sample of ETGs up to z=1, extracted from the zCOSMOS survey with a new selection criterion. The color distributions and the slopes of the color-mass and color-environment relations are studied, finding a strong dependence on mass and a minor dependence on environment. The spectral analysis performed on the D4000 and Hδ features gives results validating the previous analysis. The main driver of galaxy evolution is found to be the galaxy mass, the environment playing a subdominant but non negligible role. The age distribution of ETGs is also analyzed as a function of mass, providing strong evidences supporting a downsizing scenario. The possibility of setting cosmological constraints studying the age-redshift relation is studied, discussing the relative degeneracies and model dependencies. A new approach is developed, aiming to minimize the impact of systematics on the “cosmic chronometer” method. Analyzing theoretical models, it is demonstrated that the D4000 is a feature correlated almost linearly with age at fixed metallicity, depending only minorly on the models assumed or on the SFH chosen. The analysis of a SDSS sample of ETGs shows that it is possible to use the differential D4000 evolution of the galaxies to set constraints to cosmological parameters in an almost model-independent way. Values of the Hubble constant and of the dark energy EoS parameter are found, which are not only fully compatible, but also with a comparable error budget with the latest results.

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.

Investigation on the Navigation Anomaly of Pioneer 10 and 11 probes

The present work consists of the investigation of the navigation of Pioneer 10 and 11 probes becoming known as the “Pioneer Anomaly”: the trajectories followed by the spacecrafts did not match the ones retrieved with standard navigation software. Mismatching appeared as a linear drift in the Doppler data received by the spacecrafts, which has been ascribed to a constant sunward acceleration of about 8.5×10-10 m/s2. The study presented hereafter tries to find a convincing explanation to this discrepancy. The research is based on the analysis of Doppler tracking data through the ODP (Orbit Determination Program), developed by NASA/JPL. The method can be summarized as: seek for any kind of physics affecting the dynamics of the spacecraft or the propagation of radiometric data, which may have not been properly taken into account previously, and check whether or not these might rule out the anomaly. A major effort has been put to build a thermal model of the spacecrafts for predicting the force due to anisotropic thermal radiation, since this is a model not natively included in the ODP. Tracking data encompassing more than twenty years of Pioneer 10 interplanetary cruise, plus twelve years of Pioneer 11 have been analyzed in light of the results of the thermal model. Different strategies of orbit determination have been implemented, including single arc, multi arc and stochastic filters, and their performance compared. Orbital solutions have been obtained without the needing of any acceleration other than the thermal recoil one indicating it as the responsible for the observed linear drift in the Doppler residuals. As a further support to this we checked that inclusion of additional constant acceleration as does not improve the quality of orbital solutions. All the tests performed lead to the conclusion that no anomalous acceleration is acting on Pioneers spacecrafts.

Functional and Genetic characterization of new genomic islands from an E. coli strain associated with neonatal meningitis.

Enterobacteriaceae genomes evolve through mutations, rearrangements and horizontal gene transfer (HGT). The latter evolutionary pathway works through the acquisition DNA (GEI) modules of foreign origin that enhances fitness of the host to a given environment. The genome of E. coli IHE3034, a strain isolated from a case of neonatal meningitis, has recently been sequenced and its subsequent sequence analysis has predicted 18 possible GEIs, of which: 8 have not been previously described, 5 fully meet the pathogenic island definition and at least 10 that seem to be of prophagic origin. In order to study the GEI distribution of our reference strain, we screened for the presence 18 GEIs a panel of 132 strains, representative of E. coli diversity. Also, using an inverse nested PCR approach we identified 9 GEI that can form an extrachromosomal circular intermediate (CI) and their respective attachment sites (att). Further, we set up a qPCR approach that allowed us to determine the excision rates of 5 genomic islands in different growth conditions. Four islands, specific for strains appertaining to the sequence type complex 95 (STC95), have been deleted in order to assess their function in a Dictyostelium discoideum grazing assays. Overall, the distribution data presented here indicate that 16 IHE3034 GEIs are more associated to the STC95 strains. Also the functional and genetic characterization has uncovered that GEI 13, 17 and 19 are involved in the resistance to phagocitation by Dictyostelium d thus suggesting a possible role in the adaptation of the pathogen during certain stages of infection.

The search for Multiple Myeloma Stem cells: molecular characterization and self-renewal mechanisms involved in the disease persistence

The existence of Multiple Myeloma Stem cells (MMSCs)is supposed to be one of the major causes of MM drug-resistance. However, very little is known about the molecular characteristics of MMSCs, even if some studies suggested that these cells resembles the memory B cells. In order to molecularly characterize MMSCs, we isolated the 138+138- population. For each cell fraction we performed a VDJ rearrangement analysis. The complete set of aberrations were performed by SNP Array 6.0 and HG-U133 Plus 2.0 microarray analyses (Affymetrix). The VDJ rearrangement analyses confirmed the clonal relationship between the 138+ clone and the immature clone. Both BM and PBL 138+ clones showed exactly the same genomic macroalterations. In the BM and PBL 138-19+27+ cell fractions several micro-alterations (range: 1-350 Kb) unique of the memory B cells clone were highlighted. Any micro-alterations detected were located out of any genomic variants region and are presumably associated to the MM pathogenesis, as confirmed by the presence of KRAS, WWOX and XIAP genes among the amplified regions. To get insight into the biology of the clonotypic B cell population, we compared the gene expression profile of 8 MM B cells samples 5 donor B cells vs, thus showing a differential expression of 11480 probes (p-value: <0,05). Among the self-renewal mechanisms, we observed the down-regulation of Hedgehog pathway and the iperactivation of Notch and Wnt signaling. Moreover, these immature cells showed a particular phenotype correlated to resistance to proteasome inhibitors (IRE1α-XBP1: -18.0; -19.96. P<0,05). Data suggested that the MM 138+ clone might resume the end of the complex process of myelomagenesis, whereas the memory B cells have some intriguing micro-alterations and a specific transcriptional program, supporting the idea that these post germinal center cells might be involved in the transforming event that originate and sustain the neoplastic clone.

Genomic and proteomic approaches in pig meat quality research field

Pig meat and carcass quality is a complex concept determined by environmental and genetic factors concurring to the phenotypic variation in qualitative characteristics of meat (fat content, tenderness, juiciness, flavor,etc). This thesis shows the results of different investigations to study and to analyze pig meat and carcass quality focusing mainly on genomic; moreover proteomic approach has been also used. The aim was to analyze data from association studies between genes considered as candidate and meat and carcass quality in different pig breeds. The approach was used to detect new SNP in genes functionally associated to the studied traits and to confirm as candidate other genes already known. Five polymorphisms (one new SNP in Calponin 1 gene and four additional polymorphism already known in other genes) were considered on chromosome 2 (SSC2). Calponin 1 (CNN1) was associated to the studied traits and furthermore the results reported confirmed the data already known for Lactate dehydrogenase A (LDHA), Low density lipoprotein receptor (LDLR), Myogenic differentiation 1 (MYOD1) e Ubiquitin-like 5 (UBL5), in Italian Large White pigs. Using an in silico search it was possible to detect on SSC2 a new SNP of Deoxyhypusine synthase (DHPS) gene partially overlapping with WD repeat domain 83 (WDR83) gene and significant for the meat pH variation in Italian Large White (ILW) pigs. Perilipin 1 (PLIN1) mapping on chromosome 7 and Perilipin 2 (PLIN2) mapping on chromosome 1 were studied and the results obtained in Duroc breed have shown significant associations with carcass traits. Moreover a study of protein composition of porcine LD muscle, indicated an effect of temperature treatment of carcass, on proteins of the sarcoplasmic fraction and in particular on PGM1 phosphorylation. Future studies on pig meat quality should be based on the integration of different experimental approaches (genomics, proteomics, transcriptomics, etc).

Analysis of the pig genome for the identification of genomic regions affecting production traits

The aim of this work was to identify markers associated with production traits in the pig genome using different approaches. We focused the attention on Italian Large White pig breed using Genome Wide Association Studies (GWAS) and applying a selective genotyping approach to increase the power of the analyses. Furthermore, we searched the pig genome using Next Generation Sequencing (NSG) Ion Torrent Technology to combine selective genotyping approach and deep sequencing for SNP discovery. Other two studies were carried on with a different approach. Allele frequency changes for SNPs affecting candidate genes and at Genome Wide level were analysed to identify selection signatures driven by selection program during the last 20 years. This approach confirmed that a great number of markers may affect production traits and that they are captured by the classical selection programs. GWAS revealed 123 significant or suggestively significant SNP associated with Back Fat Thickenss and 229 associated with Average Daily Gain. 16 Copy Number Variant Regions resulted more frequent in lean or fat pigs and showed that different copies of those region could have a limited impact on fat. These often appear to be involved in food intake and behavior, beside affecting genes involved in metabolic pathways and their expression. By combining NGS sequencing with selective genotyping approach, new variants where discovered and at least 54 are worth to be analysed in association studies. The study of groups of pigs undergone to stringent selection showed that allele frequency of some loci can drastically change if they are close to traits that are interesting for selection schemes. These approaches could be, in future, integrated in genomic selection plans.