Candidate genes affecting fat deposition, carcass composition and meat quality traits in pigs

Pig meat quality is determined by several parameters, such as lipid content, tenderness, water-holding capacity, pH, color and flavor, that affect consumers’ acceptance and technological properties of meat. Carcass quality parameters are important for the production of fresh and dry-cure high-quality products, in particular the fat deposition and the lean cut yield. The identification of genes and markers associated with meat and carcass quality traits is of prime interest, for the possibility of improving the traits by marker-assisted selection (MAS) schemes. Therefore, the aim of this thesis was to investigate seven candidate genes for meat and carcass quality traits in pigs. In particular, we focused on genes belonging to the family of the lipid droplet coat proteins perilipins (PLIN1 and PLIN2) and to the calpain/calpastatin system (CAST, CAPN1, CAPN3, CAPNS1) and on the gene encoding for PPARg-coactivator 1A (PPARGC1A). In general, the candidate genes investigation included the protein localization, the detection of polymorphisms, the association analysis with meat and carcass traits and the analysis of the expression level, in order to assess the involvement of the gene in pork quality. Some of the analyzed genes showed effects on various pork traits that are subject to selection in genetic improvement programs, suggesting a possible involvement of the genes in controlling the traits variability. In particular, significant association results have been obtained for PLIN2, CAST and PPARGC1A genes, that are worthwhile of further validation. The obtained results contribute to a better understanding of biological mechanisms important for pig production as well as for a possible use of pig as animal model for studies regarding obesity in humans.

Nuclear shape instability caused by lamin A deregulation promotes invasiveness in pediatric bone sarcomas: from nucleo-cytoskeleton dynamics to novel therapeutic opportunities

Osteosarcoma (OS) and Ewing sarcoma (EWS) are the two most frequent primary bone tumors, in which metastases remain the most relevant adverse prognostic factor. Lamin A is the main constituent of the nuclear lamina, with a fundamental role in maintaining the connection between nucleus and cytoskeleton (through LINC complex proteins interactions), and its alterations can be implicated in tumor progression. We investigated how nucleo-cytoskeleton dynamics is influenced by lamin A modulation in OS and EWS, demonstrating that both these cancer models had low levels of lamin A, which are linked to a significantly more marked nuclear misshaping. In our in vitro studies, reduced levels of lamin A promoted migratory abilities in these tumors. Moreover, these findings were corroborated by gene expression analyses on EWS patient samples, showing that LMNA levels were significantly lower in metastatic lesions compared to primary tumors and that patients with low LMNA had a significant worse overall survival. We also found that LMNA expression significantly impaired EWS metastases formation in vivo. We demonstrated that low lamin A expression was linked to a severe mislocalization of LINC complex proteins, thus disrupting nucleo-cytoskeleton interactions, with a corresponding gain in malignant properties, which resulted in increased invasiveness. Lamin A overexpression or its accumulation by a statin-based pharmacological treatment allowed us to reconstitute a functional nucleo-cytoskeleton interplay, which resulted in significant downmodulation of ROCK2 and YAP, two crucial drivers of EWS aggressiveness. Our study demonstrated that lamin A is a favorable mediator of nuclear shape stability in bone sarcomas, and its modulation rescues LINC complex protein localization and regulates mechano-signaling pathways, thus promoting a less aggressive cancer phenotype. We also identified statins, already employed in clinical practice, as a tool capable to increase lamin A levels, and to reconstitute functional nucleo-cytoskeletal dynamics, resulting in reduced cellular migration.

Regulation of SRC-3 localization and dynamics by phosphorylation during ERα-dependent transcriptional activation

Transcription is controlled by promoter-selective transcriptional factors (TFs), which bind to cis-regulatory enhancers elements, termed hormone response elements (HREs), in a specific subset of genes. Regulation by these factors involves either the recruitment of coactivators or corepressors and direct interaction with the basal transcriptional machinery (1). Hormone-activated nuclear receptors (NRs) are well characterized transcriptional factors (2) that bind to the promoters of their target genes and recruit primary and secondary coactivator proteins which possess many enzymatic activities required for gene expression (1,3,4). In the present study, using single-cell high-resolution fluorescent microscopy and high throughput microscopy (HTM) coupled to computational imaging analysis, we investigated transcriptional regulation controlled by the estrogen receptor alpha (ERalpha), in terms of large scale chromatin remodeling and interaction with the associated coactivator SRC-3 (Steroid Receptor Coactivator-3), a member of p160 family (28) primary coactivators. ERalpha is a steroid-dependent transcriptional factor (16) that belongs to the NRs superfamily (2,3) and, in response to the hormone 17-ß estradiol (E2), regulates transcription of distinct target genes involved in development, puberty, and homeostasis (8,16). ERalpha spends most of its lifetime in the nucleus and undergoes a rapid (within minutes) intranuclear redistribution following the addition of either agonist or antagonist (17,18,19). We designed a HeLa cell line (PRL-HeLa), engineered with a chromosomeintegrated reporter gene array (PRL-array) containing multicopy hormone response-binding elements for ERalpha that are derived from the physiological enhancer/promoter region of the prolactin gene. Following GFP-ER transfection of PRL-HeLa cells, we were able to observe in situ ligand dependent (i) recruitment to the array of the receptor and associated coregulators, (ii) chromatin remodeling, and (iii) direct transcriptional readout of the reporter gene. Addition of E2 causes a visible opening (decondensation) of the PRL-array, colocalization of RNA Polymerase II, and transcriptional readout of the reporter gene, detected by mRNA FISH. On the contrary, when cells were treated with an ERalpha antagonist (Tamoxifen or ICI), a dramatic condensation of the PRL-array was observed, displacement of RNA Polymerase II, and complete decreasing in the transcriptional FISH signal. All p160 family coactivators (28) colocalize with ERalpha at the PRL-array. Steroid Receptor Coactivator-3 (SRC-3/AIB1/ACTR/pCIP/RAC3/TRAM1) is a p160 family member and a known oncogenic protein (4,34). SRC-3 is regulated by a variety of posttranslational modifications, including methylation, phosphorylation, acetylation, ubiquitination and sumoylation (4,35). These events have been shown to be important for its interaction with other coactivator proteins and NRs and for its oncogenic potential (37,39). A number of extracellular signaling molecules, like steroid hormones, growth factors and cytokines, induce SRC-3 phosphorylation (40). These actions are mediated by a wide range of kinases, including extracellular-regulated kinase 1 and 2 (ERK1-2), c-Jun N-terminal kinase, p38 MAPK, and IkB kinases (IKKs) (41,42,43). Here, we report SRC-3 to be a nucleocytoplasmic shuttling protein, whose cellular localization is regulated by phosphorylation and interaction with ERalpha. Using a combination of high throughput and fluorescence microscopy, we show that both chemical inhibition (with U0126) and siRNA downregulation of the MAP/ERK1/2 kinase (MEK1/2) pathway induce a cytoplasmic shift in SRC-3 localization, whereas stimulation by EGF signaling enhances its nuclear localization by inducing phosphorylation at T24, S857, and S860, known partecipants in the regulation of SRC-3 activity (39). Accordingly, the cytoplasmic localization of a non-phosphorylatable SRC-3 mutant further supports these results. In the presence of ERalpha, U0126 also dramatically reduces: hormone-dependent colocalization of ERalpha and SRC-3 in the nucleus; formation of ER-SRC-3 coimmunoprecipitation complex in cell lysates; localization of SRC-3 at the ER-targeted prolactin promoter array (PRL-array) and transcriptional activity. Finally, we show that SRC-3 can also function as a cotransporter, facilitating the nuclear-cytoplasmic shuttling of estrogen receptor. While a wealth of studies have revealed the molecular functions of NRs and coregulators, there is a paucity of data on how these functions are spatiotemporally organized in the cellular context. Technically and conceptually, our findings have a new impact upon evaluating gene transcriptional control and mechanisms of action of gene regulators.

Expression and cellular localization of Copper transporter 2 (Ctr2) in Mus musculus

The Ctr family is an essential part of the copper homeostasis machinery and its members share sequence homology and structural and functional features. Higher eukaryotes express two members of this family Ctr1 and Ctr2. Numerous structural and functional studies are available for Ctr1, the only high affinity Cu(I) transporter thus far identified. Ctr1 holigotrimers mediate cellular copper uptake and this protein was demonstrated to be essential for embryonic development and to play a crucial role in dietary copper acquisition. Instead very little is known about Ctr2, it bears structural homology to the yeast vacuolar copper transporter, which mediates mobilization of vacuolar copper stores. Recent studies using over-expressed epitope-tagged forms of human Ctr2 suggested a function as a low affinity copper transporter that can mediate either copper uptake from the extracellular environment or mobilization of lysosomal copper stores. Using an antibody that recognizes endogenous mouse Ctr2, we studied the expression and localization of endogenous mouse Ctr2 in cell culture and in mouse models to understand its regulation and function in copper homeostasis. By immunoblot we observed a regulation of mCtr2 protein levels in a copper and Ctr1 dependent way. Our observations in cells and transgenic mice suggest that lack of Ctr1 induces a strong downregulation of Ctr2 probably by a post-translational mechanism. By indirect immunofluorescence we observed an exclusive intracellular localization in a perinuclear compartment and no co-localization with lysosomal markers. Immunofluorescence experiments in Ctr1 null cells, supported by sequence analysis, suggest that lysosomes may play a role in mCtr2 biology not as resident compartment, but as a degradation site. In appendix a LC-mass method for analysis of algal biotoxins belonging to the family of PsP (paralytic shellfish poisoning) is described.

Detection and localization of GLUTs in spermatozoa from different domestic species

Sperm cells need hexoses as a substrate for their function, for both the maintenance of membrane homeostasis and the movement of the tail. These cells have a peculiar metabolism that has not yet been fully understood, but it is clear that they obtain energy from hexoses through glycolisis and/or oxidative phosphorylation. Spermatozoa are in contact with different external environments, beginning from the testicular and epididymal fluid, passing to the seminal plasma and finally to the female genital tract fluids; in addition, with the spread of reproductive biotechnologies, sperm cells are diluted and stored in various media, containing different energetic substrates. To utilize these energetic sources, sperm cells, as other eukaryotic cells, have a well-constructed protein system, that is mainly represented by the GLUT family proteins. These transporters have a membrane-spanning α-helix structure and work as an enzymatic pump that permit a fast gradient dependent passage of sugar molecules through the lipidic bilayer of sperm membrane. Many GLUTs have been studied in man, bull and rat spermatozoa; the presence of some GLUTs has been also demonstrated in boar and dog spermatozoa. The aims of the present study were - to determine the presence of GLUTs 1, 2, 3, 4 and 5 in boar, horse, dog and donkey spermatozoa and to describe their localization; - to study eventual changes in GLUTs location after capacitation and acrosome reaction in boar, stallion and dog spermatozoa; - to determine possible changes in GLUTs localization after capacitation induced by insulin and IGF stimulation in boar spermatozoa; - to evaluate changes in GLUTs localization after flow-cytometric sex sorting in boar sperm cells. GLUTs 1, 2, 3 and 5 presence and localization have been demonstrated in boar, stallion, dog and donkey spermatozoa by western blotting and immunofluorescence analysis; a relocation in GLUTs after capacitation has been observed only in dog sperm cells, while no changes have been observed in the other species examined. As for boar, the stimulation of the capacitation with insulin and IGF didn’t cause any change in GLUTs localization, as well as for the flow cytometric sorting procedure. In conclusion, this study confirms the presence of GLUTs 1, 2 ,3 and 5 in boar, dog, stallion and donkey spermatozoa, while GLUT 4 seems to be absent, as a confirmation of other studies. Only in dog sperm cells capacitating conditions induce a change in GLUTs distribution, even if the physiological role of these changes should be deepened.

Reverse genetic studies of Benyvirus - Polymyxa betae molecular interaction: Role of the RNA4-encoded protein in virus transmission

Beet necrotic yellow vein virus (BNYVV), the leading infectious agent that affects sugar beet, is included within viruses transmitted through the soil from plasmodiophorid as Polymyxa betae. BNYVV is the causal agent of Rhizomania, which induces abnormal rootlet proliferation and is widespread in the sugar beet growing areas in Europe, Asia and America; for review see (Peltier et al., 2008). In this latter continent, Beet soil-borne mosaic virus (BSBMV) has been identified (Lee et al., 2001) and belongs to the benyvirus genus together with BNYVV, both vectored by P. betae. BSBMV is widely distributed only in the United States and it has not been reported yet in others countries. It was first identified in Texas as a sugar beet virus morphologically similar but serologically distinct to BNYVV. Subsequent sequence analysis of BSBMV RNAs evidenced similar genomic organization to that of BNYVV but sufficient molecular differences to distinct BSBMV and BNYVV in two different species (Rush et al., 2003). Benyviruses field isolates usually consist of four RNA species but some BNYVV isolates contain a fifth RNA. RNAs -1 contains a single long ORF encoding polypeptide that shares amino acid homology with known viral RNA-dependent RNA polymerases (RdRp) and helicases. RNAs -2 contains six ORFs: capsid protein (CP), one readthrough protein, triple gene block proteins (TGB) that are required for cell-to-cell virus movement and the sixth 14 kDa ORF is a post-translation gene silencing suppressor. RNAs -3 is involved on disease symptoms and is essential for virus systemic movement. BSBMV RNA-3 can be trans-replicated, trans-encapsidated by the BNYVV helper strain (RNA-1 and -2) (Ratti et al., 2009). BNYVV RNA-4 encoded one 31 kDa protein and is essential for vector interactions and virus transmission by P. betae (Rahim et al., 2007). BNYVV RNA-5 encoded 26 kDa protein that improve virus infections and accumulation in the hosts. We are interest on BSBMV effect on Rhizomania studies using powerful tools as full-length infectious cDNA clones. B-type full-length infectious cDNA clones are available (Quillet et al., 1989) as well as A/P-type RNA-3, -4 and -5 from BNYVV (unpublished). A-type BNYVV full-length clones are also available, but RNA-1 cDNA clone still need to be modified. During the PhD program, we start production of BSBMV full-length cDNA clones and we investigate molecular interactions between plant and Benyviruses exploiting biological, epidemiological and molecular similarities/divergences between BSBMV and BNYVV. During my PhD researchrs we obtained full length infectious cDNA clones of BSBMV RNA-1 and -2 and we demonstrate that they transcripts are replicated and packaged in planta and able to substitute BNYVV RNA-1 or RNA-2 in a chimeric viral progeny (BSBMV RNA-1 + BNYVV RNA-2 or BNYVV RNA-1 + BSBMV RNA-2). During BSBMV full-length cDNA clones production, unexpected 1,730 nts long form of BSBMV RNA-4 has been detected from sugar beet roots grown on BSBMV infected soil. Sequence analysis of the new BSBMV RNA-4 form revealed high identity (~100%) with published version of BSBMV RNA-4 sequence (NC_003508) between nucleotides 1-608 and 1,138-1,730, however the new form shows 528 additionally nucleotides between positions 608-1,138 (FJ424610). Two putative ORFs has been identified, the first one (nucleotides 383 to 1,234), encode a protein with predicted mass of 32 kDa (p32) and the second one (nucleotides 885 to 1,244) express an expected product of 13 kDa (p13). As for BSBMV RNA-3 (Ratti et al., 2009), full-length BSBMV RNA-4 cDNA clone permitted to obtain infectious transcripts that BNYVV viral machinery (Stras12) is able to replicate and to encapsidate in planta. Moreover, we demonstrated that BSBMV RNA-4 can substitute BNYVV RNA-4 for an efficient transmission through the vector P. betae in Beta vulgaris plants, demonstrating a very high correlation between BNYVV and BSBMV. At the same time, using BNYVV helper strain, we studied BSBMV RNA-4’s protein expression in planta. We associated a local necrotic lesions phenotype to the p32 protein expression onto mechanically inoculated C. quinoa. Flag or GFP-tagged sequences of p32 and p13 have been expressed in viral context, using Rep3 replicons, based on BNYVV RNA-3. Western blot analyses of local lesions contents, using FLAG-specific antibody, revealed a high molecular weight protein, which suggest either a strong interaction of BSBMV RNA4’s protein with host protein(s) or post translational modifications. GFP-fusion sequences permitted the subcellular localization of BSBMV RNA4’s proteins. Moreover we demonstrated the absence of self-activation domains on p32 by yeast two hybrid system approaches. We also confirmed that p32 protein is essential for virus transmission by P. betae using BNYVV helper strain and BNYVV RNA-3 and we investigated its role by the use of different deleted forms of p32 protein. Serial mechanical inoculation of wild-type BSBMV on C. quinoa plants were performed every 7 days. Deleted form of BSBMV RNA-4 (1298 bp) appeared after 14 passages and its sequence analysis shows deletion of 433 nucleotides between positions 611 and 1044 of RNA-4 new form. We demonstrated that this deleted form can’t support transmission by P. betae using BNYVV helper strain and BNYVV RNA-3, moreover we confirmed our hypothesis that BSBMV RNA-4 described by Lee et al. (2001) is a deleted form. Interesting after 21 passages we identifed one chimeric form of BSBMV RNA-4 and BSBMV RNA-3 (1146 bp). Two putative ORFs has been identified on its sequence, the first one (nucleotides 383 to 562), encode a protein with predicted mass of 7 kDa (p7), corresponding to the N-terminal of p32 protein encoded by BSBMV RNA-4; the second one (nucleotides 562 to 789) express an expected product of 9 kDa (p9) corresponding to the C-terminal of p29 encoded by BSBMV RNA-3. Results obtained by our research in this topic opened new research lines that our laboratories will develop in a closely future. In particular BSBMV p32 and its mutated forms will be used to identify factors, as host or vector protein(s), involved in the virus transmission through P. betae. The new results could allow selection or production of sugar beet plants able to prevent virus transmission then able to reduce viral inoculum in the soil.