177,300 research outputs found
Role of the RNA-binding protein Sam68 in prostate cancer cell survival and proliferation
No full textIl tumore prostatico si sviluppa come una iper-proliferazione dipendente dagli androgeni delle cellule epiteliali ghiandolari e viene inizialmente affrontato con una terapia anti-androgenica. Tuttavia, dopo una regressione iniziale, il tumore si evolve in una forma più aggressiva indipendente dagli androgeni per cui ad oggi non è stata ancora trovata una cura (Grossmann et al., 2001). Nel nostro laboratorio è stata precedentemente descritta l’attivazione della tirosin chinasi Src in un gruppo di tumori prostatici avanzati, correlata alla fosforilazione in tirosina della RNA-binding protein Sam68 (Pronetto et al., 2004) appartenente alla famiglia STAR (Signal transduction and RNA metabolism), coinvolta nello splicing e nel processamento dei pre-mRNA (Lukong and Richard, 2003).
Da qui abbiamo analizzato l’espressione e la funzione di Sam68 in cellule tumorali. Abbiamo osservato che, nei pazienti affetti da PCa, Sam68 è up-regolata sia a livello di mRNA che a livello di proteina. La down-regolazione di Sam68 tramite RNAi o interferire con la sua funzione in vivo con una proteina chimerica (GFP-Sam68GSG ) determinano un rallentamento della proliferazione di cellule tumorali prostatiche e le rendono più suscettibili all’apoptosi indotta da agenti chemoterapici. Questi dati mostrano quindi che l’espressione di Sam68 favorisce la proliferazione delle cellule tumorali di prostata e la sopravvivenza ad agenti chemoterapici (Busà et al., 2007).
Ci siamo poi concentrati sullo studio del ruolo di Sam68 in questi eventi a livello molecolare. Abbiamo osservato che nelle cellule PC3, una linea di tumore prostatico non responsiva agli androgeni, in seguito a trattamento con l’agente chemoterapico mitoxantrone Sam68 rilocalizza all’interno di granuli nucleari. Abbiamo caratterizzato questi granuli nucleari ed abbiamo visto che in essi Sam68 colocalizza con diverse RNA-binding protein, sia appartenenti alla famiglia SR (SC35 e ASF/SF2) sia coinvolte nella risposta cellulare allo stress(hnRNP A1 e TIA1) (Guil et al., 2006). Sam68 si accumula anche in granuli citoplasmatici in cui co-localizza sia con hnRNP A1 che con TIA1, confermando si tratti dei cosiddetti stress granules (SGs). Questi dati suggeriscono che Sam68 faccia parte di una risposta cellulare allo stress “RNA-mediata”. Inoltre, poiché questa proteina è in grado di legare gli mRNA e di mediare lo splicing alternativo di pre-mRNA, abbiamo cercato di identificare i target modulati dal trattamento con il chemoterapico. In particolare ci siamo concentrati sullo splicing alternativo di un target già noto di Sam68, CD44 (Matter et al., 2002). Siamo andati ad analizzare lo splicing alternativo del pre-mRNA di CD44 in seguito a una dose-risposta con il mitoxantrone ed abbiamo riscontrato delle variazioni di splicing di alcuni esoni variabili, in particolare per v5 e v6, che sono noti essere regolati da Sam68 (Matter et al., 2002; Cheng and Sharp, 2006). Per valutare se le differenze osservate sono dovute alla rilocalizzazione di Sam68 effettueremo trattamenti con il chemoterapico su cellule silenziate per Sam68. Abbiamo individuato le vie biochimiche e di trasduzione del segnale che si accendono in risposta al trattamento con il mitoxantrone, la via del DNA damage di ATM e la via delle MAPkinasi indotta da stress di JNK1/2 e p38. Attraverso l’uso di inibitori specifici, per ATM, p38 e JNK, abbiamo osservato che queste vie non sono necessarie per la rilocalizzazione di Sam68. E ‘ dunque possibile che cambiamenti di conformazione della cromatina stimolino l’accumulo si Sam68 ed altri splicing factors nei granuli nucleari.
Infine, alcune evidenze emerse nel corso dei nostri studi suggeriscono un nuovo ruolo di Sam68 nel metabolismo degli rRNA. In un esperimento di co-immunoprecipitazione per Sam68, tra le proteine lin grado di interagire con Sam68 abbiamo identificato Nucleolina, una proteina nucleolare coinvolta nel metabolismo del rRNA (Rickards et al., 2007). Abbiamo confermato quest’interazione e mappato la regione di legame nel dominio carbossi-terminale di Sam68. Inoltre, in un esperimento di co-immunoprecipitazione per Sam68 ed RNA, abbiamo identificato il 18S rRNA tra gli RNA legati da questa proteina. Abbiamo inoltre osservato, attraverso esperimenti di FISH confermati poi da real time PCR, che la down-regolazione di Sam68 determina un aumento significativo dei livelli del pre-rRNA in confronto alle cellule di controllo. Infine esperimenti di ChIP hanno dimostrato che Sam68 è in grado di legare il rDNA a cavallo della regione codificante per il 18S rRNA. Questi risultati suggeriscono un nuovo ruolo di Sam68 nel metabolismo del pre-rRNA.
References:
Busà R, Paronetto MP, Farini D, Pierantozzi E, Botti F, Angelini DF, Attisani F, Vespasiani G, Sette C., Oncogene 2007 26(30):4372-82.
Cheng C, Sharp PA. (2006). Regulation of CD44 alternative splicing by SRm160 and its potential role in tumor cell invasion. Mol Cell Biol. 26(1):362-70.
Grossmann ME, Tindall DJ (2001). Androgen receptor signaling in androgen-refractory prostate cancer. J Natl Cancer Inst. 93:1687-97;
Guil S, Long JC, Cáceres JF. (2006). hnRNP A1 relocalization to the stress granules reflects a role in the stress response. Mol Cell Biol. 26(15):5744-58.
Lukong KE, Richard S (2003). Sam68, the KH domain-containing superSTAR. Bioch. Biophys. Acta 1653: 73-86.
Matter N, Herrlich P, Konig H (2002). Signal-dependent regulation of splicing via phosphorylation of Sam68. Nature 420:691-695.
Paronetto MP, Farini D, Sammarco I, Maturo G, Vespasiani G, Geremia R et al (2004). Expression of a truncated form of the c-Kit tyrosine kinase receptor and activation of Src kinase in human prostatic cancer. Am. J. Path. 164:1243-1251;
Rickards B, Flint SJ, Cole MD, LeRoy G. (2007). Nucleolin is required for RNA polymerase I transcription in vivo. Mol Cell Biol. 27(3):937-48.Prostate carcinoma (PCa) is one of the main causes of death in the western male population. Although initially controlled by anti-androgenic therapies, PCa often evolves to become androgen-insensitive and highly metastatic. A predominant role in the development of androgen-refractoriness is played by the upregulation of signal transduction pathways that allow prostate cancer cells to autonomously produce their own requirements of growth factors and nutrients (Grossmann et al., 2001).
The tyrosine kinase Src is frequently activated in advanced human prostate carcinomas and in our laboratory we have observed that its activation correlates with tyrosine phosphorylation of the RNA-binding protein Sam68 (Paronetto et al., 2004), belonging to the STAR family (Signal transduction and RNA metabolism) and involved in RNA metabolism.
In the first part of this PhD Thesis, we have investigated the expression and function of Sam68 in human prostate cancer cells. We observed that Sam68 is up-regulated both at protein and mRNA levels in patients affected by PCa. Moreover, it was observed that down-regulation of Sam68 by RNAi in LNCaP prostate cancer cells delayed cell cycle progression, reduced the proliferation rate and sensitized cells to apoptosis induced by DNA-damaging agents. Microarray analyses revealed that a subset of genes involved in proliferation and apoptosis were altered when Sam68 was knocked down in LNCaP cells. Finally, stable cell lines expressing a truncated GFP-Sam68GSG protein, that interacts with endogenous Sam68 affecting its activity, displayed reduced growth rates and higher sensitivity to cisplatin-induced apoptosis, resembling down-regulation of Sam68 by RNAi. Together, these results indicate that Sam68 expression supports prostate cancer cells proliferation and survival to cytotoxic agents (Busà et al., 2007).
Stemming from this evidence, we then aimed to investigate the role played by Sam68 in the response to genotoxic drugs such as mitoxantrone (MTX), a topoisomerase II inhibitor.We observed that MTX caused a subcellular re-localization of Sam68 from nucleoplasm to nuclear granules. Co-staining experiments indicated that Sam68-positive nuclear granules are sites of accumulation of several RNA-binding proteins involved in alternative splicing, such as SR proteins like SC35 and ASF/SF2, and TIA-1 and hnRNP A1, involved in cellular stress responses to various stimuli (Guil et al., 2006). Sam68 also accumulated in cytoplasmic granules that were also co-stained with hnRNP A1 and TIA-1, suggesting that these structures are the well described cytoplasmic stress granules (SGs). These data strongly suggest that Sam68 is part of a RNA-mediated stress response of the cell. Thus, we have begun to investigate whether changes in subcellular localization of Sam68 induced by genotoxic drugs affect alternative splicing of Sam68 target mRNAs, such as CD44 (Matter et al., 2002). Preliminary experiments have shown that MTX treatment in PC3 cells induces changes in alternative splicing of CD44 pre-mRNA. In particular, inclusion of variable exons v5 and v6, known to be regulated by Sam68 (Matter et al., 2002; Cheng and Sharp, 2006), was stimulated. We are current extending these studies to determine whether downregulation of Sam68 by RNAi affects these modifications of CD44 alternative splicing caused by MTX
Since Sam68 is known to link signal transduction pathways to RNA metabolism (Lukong and Richard, 2003), we asked whether changes in Sam68 subcellular localization induced by MTX are determined by activation of specific signal transduction pathways. Our data show that although MTX triggers activation of DNA damage pathway, through ATM kinase, and stress-induced MAPKs p38 and JNK1/2 pathways, specific inhibition of these pathways did not affect the subcellular relocalization of Sam68. Thus, it is possible that direct changes in the chromatin structure or function trigger the observed accumulation of Sam68 and splicing factors in nuclear granules.
Finally, a set of observations performed during our studies implicate Sam68 in nucleolar functions. In a co-immunoprecipitation experiment aimed at the identification of Sam68-interacting proteins in LNCaP cells we found Nucleolin, a nucleolar protein involved in rRNA metabolism (Rickards et al., 2007). This interaction has been confirmed and mapped to the carboxyterminal region of Sam68 by in vitro studies. Moreover, a RNA-protein co-immunoprecipitation experiment revealed that Sam68 binds 18S rRNA These observations lead us to investigate whether Sam68 plays a role in rRNA metabolism. First, we observed by FISH analysis, and then confermed by real time PCR, that downregulation of Sam68 caused a significant increase in the levels of pre-rRNA compared with control siRNA treated cells. Moreover, ChIP assays aimed at determining the site of the association of Sam68 with rDNA in PC3 cells revealed that Sam68 binds the 18S rRNA coding region. Thus, the results presented herein strongly suggest a novel role of Sam68 in the regulation of pre-rRNA maturation. Our current studies are aimed at investigating this hypothesis further.
References:
Busà R, Paronetto MP, Farini D, Pierantozzi E, Botti F, Angelini DF, Attisani F, Vespasiani G, Sette C., Oncogene 2007 26(30):4372-82.
Cheng C, Sharp PA. (2006). Regulation of CD44 alternative splicing by SRm160 and its potential role in tumor cell invasion. Mol Cell Biol. 26(1):362-70.
Grossmann ME, Tindall DJ (2001). Androgen receptor signaling in androgen-refractory prostate cancer. J Natl Cancer Inst. 93:1687-97;
Guil S, Long JC, Cáceres JF. (2006). hnRNP A1 relocalization to the stress granules reflects a role in the stress response. Mol Cell Biol. 26(15):5744-58.
Lukong KE, Richard S (2003). Sam68, the KH domain-containing superSTAR. Bioch. Biophys. Acta 1653: 73-86.
Matter N, Herrlich P, Konig H (2002). Signal-dependent regulation of splicing via phosphorylation of Sam68. Nature 420:691-695.
Paronetto MP, Farini D, Sammarco I, Maturo G, Vespasiani G, Geremia R et al (2004). Expression of a truncated form of the c-Kit tyrosine kinase receptor and activation of Src kinase in human prostatic cancer. Am. J. Path. 164:1243-1251;
Rickards B, Flint SJ, Cole MD, LeRoy G. (2007). Nucleolin is required for RNA polymerase I transcription in vivo. Mol Cell Biol. 27(3):937-48
Il progetto d’architettura come rilettura e riscrittura
No full textAs in Italo Calvino’s description of Zaira, every place we encounter is never merely a spatial configuration, but rather a complex fabric of memories, stories, and perceptions. These are often invisible traces, sometimes forgotten, yet they continue to shape the way we inhabit urban space. The city is not only matter, but also narrative, experience, and emotional stratification: within it, every element becomes charged with meanings that connect the present to the past.
From this awareness emerged the design intervention in Alcalá de Henares. The goal was to establish an authentic dialogue with the existing urban fabric, reinterpreting its historical layers through formal choices capable of evoking rather than imitating them. Materials, geometries, and alignments became tools for a harmonious interaction with the city’s identity-bearing monuments, particularly with the Catedral Magistral de los Santos Justo y Pastor. The project thus presents itself as an act of attentive listening and reinterpretation, where new architecture arises from engagement with history, restoring meaning and continuity to an urban landscape rich in significance
Aspects of consonant cluster mutations: the case of /sr/ sequences in Italian
No full textSequences of lingual fricatives + apical trills involve conflicting configurations of the tongue-tip/dorsum and create the conditions for assimilatory changes leading to the weakening or loss of the lingual fricative before the trill. In Italian, unlike other Romance languages, no assimilation of /s/ to /r/ has been reported. The aim of this paper is to explore the phonetic characteristics of /sr/ clusters in Italian and understand what prevents the assimilation of the lingual fricative to the apical trill. The hypothesis tested is that this may be due to the insertion of an epenthetic consonant or vowel at the release of the fricative impeding the gestural overlap between the fricative and the trill. To this purpose, the target sequences /VzrV/, /Vz(#)rV/, /Vs##rV/, and the control sequences /Vr:V/, /Vr##rV/, /V#zdrV/, /V#strV/ were embedded in real words and read by Northern and Southern Italian speakers. The acoustic characteristics of the segments in the sequences were measured. The results show that the fricative is maintained in Italian /sr/ sequences, and that an epenthetic vowel occurs after the obstruent in the test and control sequences. In addition, in the /sr/ sequences, the realization of the rhotic as a tap followed by a burst or period of frication may provide the cues for an epenthetic voiced stop at the transition between the /s/ and the /r/. The data also provide evidence for the presence of an actual stop occurring between /s/ and /r/, which parallels historical patterns from Latin into Romance
Innesti ossei neovascolarizzati. Studio sperimentale preliminare.
No full textGli innesti ossei vascolarizzati sono oggi procedure standardizzate in chirurgia ricostruttiva ma presentano alcuni svantaggi: la morbidità del sito donatore, il numero limitato di zone donatrici "naturali", la tecnica complessa. La neo-angiogenesi è un fenomeno di comune osservazione nei processi riparativi e proliferativi dei tessuti. Nel presente studio abbiamo voluto testare la possibilità di creare un innesto osseo "neo-vascolarizzato" utilizzando una procedura di impianto vascolare nel tessuto osseo del coniglio. Lo studio è stato condotto su 16 conigli New Zealand adulti. Il prelievo di due frammenti ossei di dimensioni mm 10x7x7 è stato effettuato a livello delle ali iliache di ogni animale. L'impianto del peduncolo vascolare femorale superficiale è stato effettuato in uno dei due frammenti. Il frammento sottoposto ad impianto vascolare è stato avvolto in un involucro di silicone e posizionato in una tasca sottocutanea a livello della coscia destra. Sul lato sinistro il secondo frammento è stato invece avvolto nel foglietto di silicone e posizionato in sede sottocutanea senza eseguire impianto vascolare. Abbiamo suddiviso gli animali in due gruppi di 8 unità ciascuno: nel gruppo I l'espianto è avvenuto dopo 4 settimane, nel gruppo II a otto settimane. Lo studio è stato condotto mediante iniezione di tetraciclina 72h prima dell'espianto per valutare la deposizione di nuovo tessuto osseo. Per studiare la neo-vascolarizzazione, in alcuni frammenti di ciascun gruppo è stata effettuata iniezione di inchiostro colloidale (mediante cateterizzazione dell'arteria centrale) prima della inclusione in PMMA. Lo studio istologico ha dimostrato nei frammenti a 8 settimane, la formazione di una ricca rete di vasi neoformati con cellule ossee paragonabili a quelle del tessuto normale. Anche la marcatura con tetraciclina ha evidenziato un migliore trofismo osseo a 8 settimane con deposizione ossea di aspetto normale. I frammenti non vascolarizzati hanno dimostrato assenza di segni diretti ed indiretti di "vitalità" cellulare e tessutale dell'osso. La procedura di impianto vascolare si è dimostrata in grado di indurre neo-vascolarizzazione e neo-deposizione ossea in un piccolo frammento osseo. Questo modello è semplice, ripetibile ed interessante da un punto di vista biologico e riteniamo che possa avere utili ripercussioni in campo clinico ortopedico
Neovascularized bone grafts: experimental investigation
No full textVascularized bone grafts are standardized procedures in reconstructive surgery but there are some disadvantages: donor site morbidity, limited number of natural donor sites, and complex technique. In this study, we test the possibility of creating a neovascularized bone graft utilizing a vascular implantation procedure in a rabbit model. Sixteen New Zealand adult white rabbits were used. In each animal, two iliac crest bone grafts (7 x 7 x 10 mm) were harvested. Vascular implantation of the right superficial femoral Vessels was performed in one of the two grafts, which was wrapped in a silicone envelope to avoid neovascularization from the surrounding tissues and positioned in a subcutaneous pocket in the right medial thigh. On the left side, the bone block, wrapped in the silicone envelope, was buried subcutaneously without vascular implantation. The operated animals were divided into two groups: Group I included eight rabbits explanted 4 weeks postoperatively and Group II included eight rabbits explanted 8 weeks postoperatively. Tetracycline injection was performed 72 hours preexplantation to evaluate new bone formation. Selective colloidal ink injection in the axial artery was performed to investigate the neovascularization before inclusion in poly-methyl-methacrylate (PMMA). Histological examination was performed in all explanted specimens comparatively. Histological examination 8 weeks after surgery showed a marked neovascularization, with normal bone cells. Tetracycline labeling showed new bone formation with a normal pattern. In ail nonvascularized specimens, no viable cells or neovascularization and no bone formation were found. The vascular implantation procedure can induce a good neovascularization with new bone formation in a small bone graft. The possibility of neovascularization induction by the simple vascular implantation procedure has several clinical implications in reconstructive surgery
RJL1-016 - Sambaoa and Mapao of Sikini-Kaipu, Busa
No full text(1) Sambaoana Mapao of Sikini-Kaipu - Sangai Nemongo - Pina (2) Panasonic L to R - Busa - Lyandaumali - Master; [Further contextual information about the Lacey Collection is held by the Pacific Archives at the Australian National University Archives]. Language as given: Eng
Questionario europeo di consenso sui reimpianti di arto superiore
No full textQuestionario europeo di consenso sui reimpianti di arto superiore
A highly localized activation current yet widespread intercellular calcium increase in the egg of the frog, Discoglossus pictus.
No full textA highly localized activation current yet widespread intercellular calcium increase in the egg of the frog, Discoglossus pictus.
No full textThe acquisition of English L2 prosody by Italian native speakers: experimental data and pedagogical implications
No full textThis paper investigates Yes-No question intonation patterns in English L2, Italian L1, and
English L1. The aim is to test the hypothesis that L2 learners may show different
acquisition strategies for different dimensions of intonation, and particularly the
phonological and phonetic components. The study analyses the nuclear intonation
contours of 4 target English words and 4 comparable Italian words consisting of sonorant
segments, stressed on the semi-final or final syllable, and occurring in Yes-No questions
in sentence-final position (e.g., Will you attend the memorial?, Hai sentito la Melania?).
The words were contained in mini-dialogues of question-answer pairs, and read 5 times
by 4 Italian speakers (Padova area, North-East Italy) and 3 English female speakers
(London area, UK). The results show that: 1) different intonation patterns may be used to
realize the same grammatical function; 2) different developmental processes are at work,
including transfer of L1 categories and the acquisition of L2 phonological categories.
These results suggest that the phonetic dimension of L2 intonation may be more difficult
to learn than the phonological one
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