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.

Effetti dell'integrazione visuo-acustica in pazienti con disturbo di campo visivo

Human brain is provided with a flexible audio-visual system, which interprets and guides responses to external events according to spatial alignment, temporal synchronization and effectiveness of unimodal signals. The aim of the present thesis was to explore the possibility that such a system might represent the neural correlate of sensory compensation after a damage to one sensory pathway. To this purpose, three experimental studies have been conducted, which addressed the immediate, short-term and long-term effects of audio-visual integration on patients with Visual Field Defect (VFD). Experiment 1 investigated whether the integration of stimuli from different modalities (cross-modal) and from the same modality (within-modal) have a different, immediate effect on localization behaviour. Patients had to localize modality-specific stimuli (visual or auditory), cross-modal stimulus pairs (visual-auditory) and within-modal stimulus pairs (visual-visual). Results showed that cross-modal stimuli evoked a greater improvement than within modal stimuli, consistent with a Bayesian explanation. Moreover, even when visual processing was impaired, cross-modal stimuli improved performance in an optimal fashion. These findings support the hypothesis that the improvement derived from multisensory integration is not attributable to simple target redundancy, and prove that optimal integration of cross-modal signals occurs in processing stage which are not consciously accessible. Experiment 2 examined the possibility to induce a short term improvement of localization performance without an explicit knowledge of visual stimulus. Patients with VFD and patients with neglect had to localize weak sounds before and after a brief exposure to a passive cross-modal stimulation, which comprised spatially disparate or spatially coincident audio-visual stimuli. After exposure to spatially disparate stimuli in the affected field, only patients with neglect exhibited a shifts of auditory localization toward the visual attractor (the so called Ventriloquism After-Effect). In contrast, after adaptation to spatially coincident stimuli, both neglect and hemianopic patients exhibited a significant improvement of auditory localization, proving the occurrence of After Effect for multisensory enhancement. These results suggest the presence of two distinct recalibration mechanisms, each mediated by a different neural route: a geniculo-striate circuit and a colliculus-extrastriate circuit respectively. Finally, Experiment 3 verified whether a systematic audio-visual stimulation could exert a long-lasting effect on patients’ oculomotor behaviour. Eye movements responses during a visual search task and a reading task were studied before and after visual (control) or audio-visual (experimental) training, in a group of twelve patients with VFD and twelve controls subjects. Results showed that prior to treatment, patients’ performance was significantly different from that of controls in relation to fixations and saccade parameters; after audiovisual training, all patients reported an improvement in ocular exploration characterized by fewer fixations and refixations, quicker and larger saccades, and reduced scanpath length. Similarly, reading parameters were significantly affected by the training, with respect to specific impairments observed in left and right hemisphere–damaged patients. The present findings provide evidence that a systematic audio-visual stimulation may encourage a more organized pattern of visual exploration with long lasting effects. In conclusion, results from these studies clearly demonstrate that the beneficial effects of audio-visual integration can be retained in absence of explicit processing of visual stimulus. Surprisingly, an improvement of spatial orienting can be obtained not only when a on-line response is required, but also after either a brief or a long adaptation to audio-visual stimulus pairs, so suggesting the maintenance of mechanisms subserving cross-modal perceptual learning after a damage to geniculo-striate pathway. The colliculus-extrastriate pathway, which is spared in patients with VFD, seems to play a pivotal role in this sensory compensation.

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.

Cooperative Wireless Networks for Localization

This thesis adresses the problem of localization, and analyzes its crucial aspects, within the context of cooperative WSNs. The three main issues discussed in the following are: network synchronization, position estimate and tracking. Time synchronization is a fundamental requirement for every network. In this context, a new approach based on the estimation theory is proposed to evaluate the ultimate performance limit in network time synchronization. In particular the lower bound on the variance of the average synchronization error in a fully connected network is derived by taking into account the statistical characterization of the Message Delivering Time (MDT) . Sensor network localization algorithms estimate the locations of sensors with initially unknown location information by using knowledge of the absolute positions of a few sensors and inter-sensor measurements such as distance and bearing measurements. Concerning this issue, i.e. the position estimate problem, two main contributions are given. The first is a new Semidefinite Programming (SDP) framework to analyze and solve the problem of flip-ambiguity that afflicts range-based network localization algorithms with incomplete ranging information. The occurrence of flip-ambiguous nodes and errors due to flip ambiguity is studied, then with this information a new SDP formulation of the localization problem is built. Finally a flip-ambiguity-robust network localization algorithm is derived and its performance is studied by Monte-Carlo simulations. The second contribution in the field of position estimate is about multihop networks. A multihop network is a network with a low degree of connectivity, in which couples of given any nodes, in order to communicate, they have to rely on one or more intermediate nodes (hops). Two new distance-based source localization algorithms, highly robust to distance overestimates, typically present in multihop networks, are presented and studied. The last point of this thesis discuss a new low-complexity tracking algorithm, inspired by the Fano’s sequential decoding algorithm for the position tracking of a user in a WLAN-based indoor localization system.

Testing future weak lensing surveys through simulations of observations

Weak lensing experiments such as the future ESA-accepted mission Euclid aim to measure cosmological parameters with unprecedented accuracy. It is important to assess the precision that can be obtained in these measurements by applying analysis software on mock images that contain many sources of noise present in the real data. In this Thesis, we show a method to perform simulations of observations, that produce realistic images of the sky according to characteristics of the instrument and of the survey. We then use these images to test the performances of the Euclid mission. In particular, we concentrate on the precision of the photometric redshift measurements, which are key data to perform cosmic shear tomography. We calculate the fraction of the total observed sample that must be discarded to reach the required level of precision, that is equal to 0.05(1+z) for a galaxy with measured redshift z, with different ancillary ground-based observations. The results highlight the importance of u-band observations, especially to discriminate between low (z < 0.5) and high (z ~ 3) redshifts, and the need for good observing sites, with seeing FWHM < 1. arcsec. We then construct an optimal filter to detect galaxy clusters through photometric catalogues of galaxies, and we test it on the COSMOS field, obtaining 27 lensing-confirmed detections. Applying this algorithm on mock Euclid data, we verify the possibility to detect clusters with mass above 10^14.2 solar masses with a low rate of false detections.