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.

Integrating new and traditional approaches for the estimate of slip-rates of active faults: examples from the Mw 6.3, 2009 L’Aquila earthquake area, Central Italy

This thesis is based on the integration of traditional and innovative approaches aimed at improving the normal faults seimogenic identification and characterization, focusing mainly on slip-rate estimate as a measure of the fault activity. The L’Aquila Mw 6.3 April 6, 2009 earthquake causative fault, namely the Paganica - San Demetrio fault system (PSDFS), was used as a test site. We developed a multidisciplinary and scale‐based strategy consisting of paleoseismological investigations, detailed geomorphological and geological field studies, as well as shallow geophysical imaging and an innovative application of physical properties measurements. We produced a detailed geomorphological and geological map of the PSDFS, defining its tectonic style, arrangement, kinematics, extent, geometry and internal complexities. The PSDFS is a 19 km-long tectonic structure, characterized by a complex structural setting and arranged in two main sectors: the Paganica sector to the NW, characterized by a narrow deformation zone, and the San Demetrio sector to SE, where the strain is accommodated by several tectonic structures, exhuming and dissecting a wide Quaternary basin, suggesting the occurrence of strain migration through time. The integration of all the fault displacement data and age constraints (radiocarbon dating, optically stimulated luminescence (OSL) and tephrochronology) helped in calculating an average Quaternary slip-rate representative for the PSDFS of 0.27 - 0.48 mm/yr. On the basis of its length (ca. 20 km) and slip per event (up to 0.8 m) we also estimated a max expected Magnitude of 6.3-6.8 for this fault. All these topics have a significant implication in terms of surface faulting hazard in the area and may contribute also to the understanding of the PSDFS seismic behavior and of the local seismic hazard.

Cellular localization and mechanism of amyloid precursor protein (APP) homodimer formation in an oxidizing environment

The amyloid precursor protein (APP) is a type I transmembrane glycoprotein, which resembles a cell surface receptor, comprising a large ectodomain, a single spanning transmembrane part and a short C-terminal, cytoplasmic domain. It belongs to a conserved gene family, with over 17 members, including also the two mammalian APP homologues proteins APLP1 and APLP2 („amyloid precursor like proteins“). APP is encoded by 19 exons, of which exons 7, 8, and 15 can be alternatively spliced to produce three major protein isoforms APP770, APP751 and APP695, reflecting the number of amino acids. The neuronal APP695 is the only isoform that lacks a Kunitz Protease Inhibitor (KPI) domain in its extracellular portion whereas the two larger, peripheral APP isoforms, contain the 57-amino-acid KPI insert. rnRecently, research effort has suggested that APP metabolism and function is thought to be influenced by homodimerization and that the oligomerization state of APP could also play a role in the pathology of Alzheimer's disease (AD), by regulating its processing and amyloid beta production. Several independent studies have shown that APP can form homodimers within the cell, driven by motifs present in the extracellular domain, as well as in the juxtamembrane (JM) and transmembrane (TM) regions of the molecule, whereby the exact molecular mechanism and the origin of dimer formation remains elusive. Therefore, we focused in our study on the actual subcellular origin of APP homodimerization within the cell, an underlying mechanism, and a possible impact on dimerization properties of its homologue APLP1. Furthermore, we analyzed homodimerization of various APP isoforms, in particular APP695, APP751 and APP770, which differ in the presence of a Kunitz-type protease inhibitor domain (KPI) in the extracellular region. In order to assess the cellular origin of dimerization under different cellular conditions, we established a mammalian cell culture model-system in CHO-K1 (chinese hamster ovary) cells, stably overexpressing human APP, harboring dilysine based organelle sorting motifs at the very C-terminus [KKAA-Endoplasmic Reticulum (ER); KKFF-Golgi]. In this study we show that APP exists as disulfide-bound, SDS-stable dimers, when it was retained in the ER, unlike when it progressed further to the cis-Golgi, due to the KKFF ER exit determinant. These stable APP complexes were isolated from cells, and analyzed by SDS–polyacrylamide gel electrophoresis under non-reducing conditions, whereas strong denaturing and reducing conditions completely converted those dimers to monomers. Our findings suggested that APP homodimer formation starts early in the secretory pathway and that the unique oxidizing environment of the ER likely promotes intermolecular disulfide bond formation between APP molecules. We particularly visualized APP dimerization employing a variety of biochemical experiments and investigated the origin of its generation by using a Bimolecular Fluorescence Complementation (BiFC) approach with split GFP-APP chimeras. Moreover, using N-terminal deletion constructs, we demonstrate that intermolecular disulfide linkage between cysteine residues, exclusively located in the extracellular E1 domain, represents another mechanism of how an APP sub-fraction can dimerize within the cell. Additionally, mutational studies revealed that cysteines at positions 98 and 105, embedded in the conserved loop region within the E1 domain, are critical for interchain disulfide bond formation. Using a pharmacological treatment approach, we show that once generated in the oxidative environment of the ER, APP dimers remain stably associated during transport, reaching the plasma membrane. In addition, we demonstrate that APP isoforms, encompassing the KPI domain, exhibit a strongly reduced ability to form cis-directed dimers in the ER, whereas trans-directed cell aggregation of Drosophila Schneider (S2)-cells was isoform independent, mediating cell-cell contacts. Thus, suggesting that steric properties of KPI-APP might be the cause for weaker cis-interaction in the ER, compared to APP695. Finally, we provide evidence that APP/APLP1 heterointeractions are likewise initiated in the ER, suggesting a similar mechanism for heterodimerization. Therefore, dynamic alterations of APP between monomeric, homodimeric, and possibly heterodimeric status could at least partially explain some of the variety in the physiological functions of APP.rn

Software tools and efficient algorithms for the feature detection, feature tracking, event localization, and visualization of large sets of atmospheric data

Data sets describing the state of the earth's atmosphere are of great importance in the atmospheric sciences. Over the last decades, the quality and sheer amount of the available data increased significantly, resulting in a rising demand for new tools capable of handling and analysing these large, multidimensional sets of atmospheric data. The interdisciplinary work presented in this thesis covers the development and the application of practical software tools and efficient algorithms from the field of computer science, aiming at the goal of enabling atmospheric scientists to analyse and to gain new insights from these large data sets. For this purpose, our tools combine novel techniques with well-established methods from different areas such as scientific visualization and data segmentation. In this thesis, three practical tools are presented. Two of these tools are software systems (Insight and IWAL) for different types of processing and interactive visualization of data, the third tool is an efficient algorithm for data segmentation implemented as part of Insight.Insight is a toolkit for the interactive, three-dimensional visualization and processing of large sets of atmospheric data, originally developed as a testing environment for the novel segmentation algorithm. It provides a dynamic system for combining at runtime data from different sources, a variety of different data processing algorithms, and several visualization techniques. Its modular architecture and flexible scripting support led to additional applications of the software, from which two examples are presented: the usage of Insight as a WMS (web map service) server, and the automatic production of a sequence of images for the visualization of cyclone simulations. The core application of Insight is the provision of the novel segmentation algorithm for the efficient detection and tracking of 3D features in large sets of atmospheric data, as well as for the precise localization of the occurring genesis, lysis, merging and splitting events. Data segmentation usually leads to a significant reduction of the size of the considered data. This enables a practical visualization of the data, statistical analyses of the features and their events, and the manual or automatic detection of interesting situations for subsequent detailed investigation. The concepts of the novel algorithm, its technical realization, and several extensions for avoiding under- and over-segmentation are discussed. As example applications, this thesis covers the setup and the results of the segmentation of upper-tropospheric jet streams and cyclones as full 3D objects. Finally, IWAL is presented, which is a web application for providing an easy interactive access to meteorological data visualizations, primarily aimed at students. As a web application, the needs to retrieve all input data sets and to install and handle complex visualization tools on a local machine are avoided. The main challenge in the provision of customizable visualizations to large numbers of simultaneous users was to find an acceptable trade-off between the available visualization options and the performance of the application. Besides the implementational details, benchmarks and the results of a user survey are presented.

Localization, web communication and seo for museum websites: The case of the Pinacoteca di Brera

La presente tesi di laurea si concentra sulla localizzazione in inglese di varie sezioni del nuovo sito web della Pinacoteca di Brera. Il progetto di localizzazione è stato contestualizzato da un lato all’interno della letteratura sulla comunicazione museale, e dall’altro sulla comunicazione web, per poter avanzare proposte di miglioramento alla luce di ricerche nel campo della SEO (Search Engine Optimization). Lo studio della comunicazione museale si è arricchito grazie all’esperienza di documentazione presso la University of Leicester (UK). La tesi mira a porre le basi per la produzione di contenuti museali adatti ad una lettura sul web, in modo da offrire non solo una traduzione ben fatta dal punto di vista linguistico e culturale, ma anche facilmente fruibile per un utente online e reperibile attraverso motori di ricerca. L’elaborato intende fornire ai musei italiani alcuni spunti di riflessione circa possibili miglioramenti delle proprie piattaforme online grazie alla localizzazione e ad un’analisi approfondita dei contenuti web secondo principi di usabilità e visibilità. Il capitolo 1 introduce la letteratura sugli studi museali, prestando particolare attenzione alla comunicazione. Il capitolo 2 fornisce una panoramica generale sul web: vengono suggerite buone pratiche di web writing, analizzate le strategie di SEO per migliorare la visibilità dei siti e delineato le principali caratteristiche del processo di localizzazione. Il capitolo 3 riunisce i due universi finora esplorati individualmente, ovvero i musei e il web, concentrandosi sulla comunicazione online dei musei e concludendo con uno schema di valutazione dei siti dei musei. Il capitolo 4 applica le strategie precedentemente discusse al caso specifico della Pinacoteca di Brera, concentrandosi sulla valutazione del sito, sulla localizzazione di alcune sezioni e sulla proposta di strategie SEO. Infine, il capitolo 5 tira le fila dell’intero lavoro mettendo in evidenza i principali risultati ottenuti.

Localization and mobility-edges in non-interacting fermionic models with disorder

Nel corso degli ultimi decenni la fisica sperimentale ha raggiunto notevoli traguardi nel campo della manipolazione di sistemi di atomi freddi, riaccendendo l'interesse della ricerca su sistemi a lungo studiati teoricamente, ma fino a poco tempo fa impossibili da realizzare sperimentalmente. Questa riaccesa attenzione ha permesso di sfruttare le moderne capacità di calcolo per studiare sistemi quantistici che ancora risultano di difficile realizzazione. In questo contesto si inserisce il rinnovato interesse per i sistemi quantistici monodimensionali caratterizzati dalla presenza di potenziale disordinato. Questi presentano proprietà di trasporto particolari e sotto particolari condizioni sono oggetto di una transizione di localizzazione. La maggior parte degli studi in questo campo rivolgono la loro attenzione a sistemi di particelle fermioniche interagenti. In questo lavoro di tesi analizziamo, invece, sistemi quantistici fermionici non interagenti, mettendo in luce quanto già noto e proponendo strumenti di analisi derivati dallo studio dei sistemi interagenti. In particolare, proponiamo un'analisi statistica dei livelli energetici e poniamo le basi per futuri studi a riguardo.

Sodium/hydrogen exchanger NHA2 in osteoclasts: subcellular localization and role in vitro and in vivo

NHA2 was recently identified as a novel sodium/hydrogen exchanger which is strongly upregulated during RANKL-induced osteoclast differentiation. Previous in vitro studies suggested that NHA2 is a mitochondrial transporter required for osteoclast differentiation and bone resorption. Due to the lack of suitable antibodies, NHA2 was studied only on RNA level thus far. To define the protein's role in osteoclasts in vitro and in vivo, we generated NHA2-deficient mice and raised several specific NHA2 antibodies. By confocal microscopy and subcellular fractionation studies, NHA2 was found to co-localize with the late endosomal and lysosomal marker LAMP1 and the V-ATPase a3 subunit, but not with mitochondrial markers. Immunofluorescence studies and surface biotinylation experiments further revealed that NHA2 was highly enriched in the plasma membrane of osteoclasts, localizing to the basolateral membrane of polarized osteoclasts. Despite strong upregulation of NHA2 during RANKL-induced osteoclast differentiation, however, structural parameters of bone, quantified by high-resolution microcomputed tomography, were not different in NHA2-deficient mice compared to wild-type littermates. In addition, in vitro RANKL stimulation of bone marrow cells isolated from wild-type and NHA2-deficient mice yielded no differences in osteoclast development and activity. Taken together, we show that NHA2 is a RANKL-induced plasmalemmal sodium/hydrogen exchanger in osteoclasts. However, our data from NHA2-deficient mice suggest that NHA2 is dispensable for osteoclast differentiation and bone resorption both in vitro and in vivo.

Immunohistochemical localization of osteoclastogenic cell mediators in feline tooth resorption and healthy teeth

Tooth resorption is among the most common and most challenging problems in feline dentistry It is a progressive disease eventually leading to tooth loss and often root replacement. The etiology of moth resorption remains obscure and to date no effective therapeutic approach is known. The present study is aimed at assessing the reliability of radiographic imaging and addressing the possible involvement of receptor activator of NF kappa B (RANK), its ligand (RANKL), and osteoprotegerin (OPG) in the process of tooth resorption. Teeth from 8 cats were investigated by means of radiographs and paraffin sections followed by immunolabeling. Six cats were diagnosed with tooth resorption based on histopathologic and radiographic findings. Samples were classified according to a four-stage diagnostic system. Radiologic assessment of tooth resorption correlated very strongly with histopathologic findings. Tooth resorption was accompanied by a strong staining with all three antibodies used, especially with anti-RANK and anti-RANKL antibodies. The presence of OPG and RANKL at the resorption site is indicative of repair attempts by fibroblasts and stromal cells. These findings should be extended by further investigations in order to elucidate the pathophysiologic processes underlying tooth resorption that might lead to prophylactic and/or therapeutic measures. J Vet Dent 27(2); 75 - 83, 2010

Cardiogoniometric parameters for detection of coronary artery disease at rest as a function of stenosis localization and distribution

Cardiogoniometry (CGM), a spatiotemporal electrocardiologic 5-lead method with automated analysis, may be useful in primary healthcare for detecting coronary artery disease (CAD) at rest. Our aim was to systematically develop a stenosis-specific parameter set for global CAD detection. In 793 consecutively admitted patients with presumed non-acute CAD, CGM data were collected prior to elective coronary angiography and analyzed retrospectively. 658 patients fulfilled the inclusion criteria, 405 had CAD verified by coronary angiography; the 253 patients with normal coronary angiograms served as the non-CAD controls. Study patients--matched for age, BMI, and gender--were angiographically assigned to 8 stenosis-specific CAD categories or to the controls. One CGM parameter possessing significance (P < .05) and the best diagnostic accuracy was matched to one CAD category. The area under the ROC curve was .80 (global CAD versus controls). A set containing 8 stenosis-specific CGM parameters described variability of R vectors and R-T angles, spatial position and potential distribution of R/T vectors, and ST/T segment alterations. Our parameter set systematically combines CAD categories into an algorithm that detects CAD globally. Prospective validation in clinical studies is ongoing.

Subcellular Localization of the Non-Structural Proteins 3C and 3CD of the Honeybee Virus Deformed Wing Virus

Apis mellifera L., the European honeybee, is a crucial pollinator of many important agricultural crops in the United States. Recently, honeybee colonies have been affected by Colony Collapse Disorder (CCD), a disorder in which the colony fails due to the disappearance of a key functional group of worker bees. Though no direct causalrelationship has been confirmed, hives that experience CCD have been shown to have a high incidence of Deformed Wing Virus (DWV), a common honeybee virus. While the genome sequence and gene-order of DWV has been analyzed fairly recently, few other studies have been performed to understand the molecular characterization of the virus.Since little is known about where DWV proteins localize in infected host cells, the objective of this project was to determine the subcellular localization of two of the important non-structural proteins that are encoded in the DWV genome. This project focused on the protein 3C, an autocatalytic protease which cleaves itself from a longer polyprotein and helps to cut all of the other proteins apart from one another so that they can become functional, and 3D, the RNA-dependent RNA polymerase (RdRp) which is critical for replication of the virus because it copies the viral genome. By tagging nested constructs containing these two proteins and tracking where they localized in living cells, this study aimed to better understand the replication of DWV and to elicit possible targetsfor further research on how to control the virus. Since DWV is a picorna-like virus, distantly related to human viruses such as polio, and picornavirus non-structural proteins aggregate at cellular membranes during viral replication, the major hypothesis was that the 3C and 3CD proteins would localize at cellular organelle membranes as well. Using confocal microscopy, both proteins were found to localize in the cytoplasm, but the 3CDprotein was found to be mostly diffuse cytoplasmic, and the 3C protein was found to localize more specifically on membranous structures just outside of the nucleus.

Design techniques and analysis of high-resolution neural recording systems targeting epilepsy focus localization

The design of a high-density neural recording system targeting epilepsy monitoring is presented. Circuit challenges and techniques are discussed to optimize the amplifier topology and the included OTA. A new platform supporting active recording devices targeting wireless and high-resolution focus localization in epilepsy diagnosis is also proposed. The post-layout simulation results of an amplifier dedicated to this application are presented. The amplifier is designed in a UMC 0.18µm CMOS technology, has an NEF of 2.19 and occupies a silicon area of 0.038 mm(2), while consuming 5.8 µW from a 1.8-V supply.